Pc131086 1.20

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has beenedited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version onlinereduces the time to publication by several weeks.
A Secreted Effector Protein of Ustilago maydis Guides MaizeLeaf Cells to Form Tumors Amey Redkar,a,1 Rafal Hoser,b Lena Schilling,a Bernd Zechmann,c Magdalena Krzymowska,bVirginia Walbot,d and Gunther Doehlemanna,e,2 a Max Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, D-35043 Marburg, Germanyb Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Polandc Baylor University, Center for Microscopy and Imaging, Waco, Texas 76798d Department of Biology, Stanford University, Stanford, California 94305e Botanical Institute and Cluster of Excellence on Plant Sciences, University of Cologne, 50674 Cologne, Germany The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues.
U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectorshave important functions in inducing tumor expansion speciﬁcally in maize leaves. Here, we present the functional characterizationof the effector See1 (Seedling efﬁcient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial fortumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel ﬂoral tissues, where maize cellproliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1),a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and humaninnate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-speciﬁc phosphorylationsite. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNAsynthesis in leaf cells. This identiﬁes See1 as a fungal effector that directly and speciﬁcally contributes to the formation of leaftumors in maize.
liberating a sooty mass of black teliospores (Brefort et al., 2009).
Among Ustilaginales, Ustilago maydis, a model organism for bio- To establish a successful infection and dampen plant defense trophic fungi (Kämper et al., 2006; Ökmen and Doehlemann, 2014), responses during colonization, plant pathogens secrete proteins has the unique ability to colonize all the aerial organs of its host and other molecules, collectively termed effectors, to various plant maize (Zea mays) and to induce the formation of plant tumors host compartments (Jones and Dangl, 2006). Effectors are key locally at sites of infection. The fungus penetrates the epidermal to the alterations of host structures and functions during infection cells and then the subepidermal cells, forming an interaction zone (Hogenhout et al., 2009). They act either in the intercellular space called the biotrophic interface in which the hyphae are encapsu- to handle the primary defense response or inside the host cell to lated by the host plasma membrane. After successful establish- execute functions such as reprogramming of the host to favor ment in leaves, the fungus grows in the mesophyll and the living infection (Doehlemann et al., 2014).
cells of the vasculature (Ökmen and Doehlemann, 2014). Prolifer- The basidiomycetous plant pathogens are highly specialized ation of host and fungal cells results in tumors, which are supported colonizers that develop biotrophic interactions. Members of the by the comprehensive reprogramming of both plant signaling and Ustilaginales, a major order of this class, invade mainly monocots, metabolism early in infection (Doehlemann et al., 2008; Horst et al., including all major cereal crops. The infection normally occurs in 2010) and alteration of the pace and pattern of host cell division.
seedlings, often immediately after mating of the compatible spor- The U. maydis genome encodes ;550 proteins that are pre- idia to form a dikaryotic ﬁlament (Kämper et al., 2006; Brefort et al., dicted to be secreted and likely function as effectors (Mueller et al., 2009). Fungal hyphae growing both intracellularly and intercellu- 2008; Djamei and Kahmann, 2012). Many potential effector genes larly colonize the host systemically and grow toward the shoot are arranged in clusters, and examination of deletion mutants re- apical meristem without inducing visible disease symptoms. Dis- vealed the importance of these genes in virulence (Kämper et al., ease symptoms become evident upon the ﬂoral transition, and the 2006; Brefort et al., 2014). So far, only a few effector genes of fungus completes sporulation within the infected inﬂorescence, U. maydis have been functionally characterized. Pep1 (Proteinessential for penetration1) is involved in penetration and the es- tablishment of initial compatibility by targeting and inhibiting the Current address: The Sainsbury Laboratory, Norwich Research Park, Norwich NR47UH, UK.
activity of the plant peroxidase POX12 (Doehlemann et al., 2009; 2 Address correspondence to Hemetsberger et al., 2012). Pit2 (Protein involved in tumors2), a The author responsible for distribution of materials integral to the ﬁndings protein essential for tissue colonization and plant defense suppres- presented in this article in accordance with the policy described in the sion, inhibits apoplastic cysteine proteases (Doehlemann et al., 2011; Instructions for Authors (is: Gunther Doehlemann Mueller et al., 2013). In addition, two translocated U. maydis ef- fectors have been analyzed. The U. maydis chorismate mutase The Plant Cell Preview, www.aspb.org ã 2015 American Society of Plant Biologists. All rights reserved.
Cmu1 rechannels chorismate metabolism in the plant cell cyto- both maize inﬂorescences and aerial vegetative tissues, such as plasm to prevent the synthesis of salicylic acid, a major defense seedling leaves (Skibbe et al., 2010). A previous analysis of signal (Djamei et al., 2011). The effector Tin2 (Tumor inducing2), U. maydis effector candidates with organ-speciﬁc expression which is part of the largest cluster of effectors in U. maydis patterns identiﬁed seven genes whose deletion resulted in a leaf- (Brefort et al., 2014), masks a ubiquitin-proteasome degradation speciﬁc reduction of tumor formation (Schilling et al., 2014), and motif in TTK1, a maize protein kinase that regulates the antho- here we investigate one of these genes (um02239, now termed cyanin biosynthetic pathway. Tin2 protects the active kinase see1) for its speciﬁc role.
against ubiquitination and thereby promotes the production of Deletion mutants of see1 (SG200Δsee1) mainly formed tumors anthocyanin in infected tissue and suppresses lignin biosyn- of 1 to 4 mm in diameter on seedling leaves at 12 DPI; these thesis, a defense pathway (Tanaka et al., 2014).
symptoms represent about half of the total tumors formed. Tu- U. maydis infects all maize aerial organs and thus interacts mors of >6 to 20 mm occurred frequently in wild-type infections, with different, developmentally distinct immature host tissues representing around 28% of the total tumors, but they occurred (Walbot and Skibbe, 2010). In a previous study, organ-speciﬁc much less frequently and were reduced in size in SG200Δsee1 transcriptomes of both the host and the pathogen were docu- infections, representing only 9% of tumors. Heavy tumors, which mented in seedlings, adult leaves, and tassels (Skibbe et al., 2010).
cause altered leaf shape or even stunted growth of infected It was hypothesized that effectors in U. maydis act in an organ- seedlings, were not observed after infection by SG200Δsee1 (Figure speciﬁc manner, a new concept now extended to anthers within 1A; ). The SG200Δsee1 mutant induces the tassels (Gao et al., 2013). A recent study showed that individual normal tumors in maize tassels, indistinguishable from the virulent effector genes of U. maydis act in speciﬁc plant organs and that progenitor strain SG200 (Figures 1A and 1B). Teliospores dissected deletion of one organ-speciﬁc effector does not hamper virulence from these tassel tumors were normal in shape and fully viable in a nontarget organ (Schilling et al., 2014). To date, however, the ). Similarly, in the maize ear, tumor formation functional basis of organ-speciﬁc effectors remains elusive.
was comparable to that in the wild type, supporting a strictly leaf- Effectors may be recognized by plant receptor proteins, which speciﬁc role of See1 in tumor induction .
in turn induce defense responses. Several plant receptor proteins Confocal microscopy showed that SG200Δsee1 hyphae initially function with the help of chaperones and cochaperones, in- colonize similarly to the progenitor strain SG200. At 3 DPI, when cluding HSP90 (heat shock protein 90), RAR1 (required for Mla12 wild-type fungal hyphae reach the leaf mesophyll and are inter- resistance), and SGT1 (suppressor of G2 allele of skp1) (Shirasu, spersed within the vasculature, mutant hyphae clustered at col- 2009; Zhang et al., 2010). SGT1 was originally identiﬁed in Sac- lapsed, highly ﬂuorescent mesophyll cells charomyces cerevisiae as an essential cell cycle protein that in- ). In addition, mutant hyphae failed to traverse from an infected teracts with Skp1p, a component of the conserved eukaryotic cell into uninfected neighboring cells; this was particularly observed Skp1/Cullin/F-box (SCF) E3 ubiquitin ligase. In yeast, Sgt1p is in bundle-sheath cells Reintroduction of required for progression through the G1/S and G2/M checkpoints the see1 gene into the U. maydis ip locus fully restored virulence, (Kitagawa et al., 1999) and is highly conserved, as its orthologs in demonstrating functional complementation of see1 and conﬁrming both animal and plant kingdoms retain the cell cycle functions that the observed growth defects reﬂected the absence of See1 (Bhavsar et al., 2013). Maturation of SGT1 as a signaling molecule (Figures 1A and 1B).
depends on phosphorylation by an upstream MAPK (Hoser et al., Transcription of see1 speciﬁcally increased during biotrophic growth of U. maydis (Figure 2A). Comparison of the temporal In this study, we present the functional characterization of and spatial proﬁles of see1 expression during successive stages the U. maydis organ-speciﬁc effector See1 (Seedling efﬁcient of tumor progression showed that see1 expression is constitu- effector1; Um02239), which is speciﬁcally required during tumor tive and then upregulated at the later stages of tumor expansion formation in seedling leaves. See1 is translocated by the fungus in maize leaves but not in tassels (Figure 2A). In maize ear tumors, into the plant cell cytoplasm and nucleus, where it interacts with see1 transcript abundance was low as in tassels at 12 DPI. At the maize homolog of SGT1 and interferes with the MAPK- this time point, see1 expression was >50-fold induced in leaves induced phosphorylation of SGT1. See1 participates in U. maydis- compared with the ﬂoral organs To gain triggered reactivation of plant DNA synthesis in maize leaves and comprehensive insight into host processes affected by see1 de- contributes to vegetative tumor formation.
letion, Agilent microarrays were used to proﬁle the transcriptomeof maize leaves at 6 DPI by SG200, SG200Δsee1, and mockcontrol infections. RNA of infected tissue was prepared from three biological replicates, analyzed by hybridization, and subjectedto data normalization and statistical analysis (see Methods fordetails). The abundances of 10,952 maize transcripts were al- See1 Is Required for the Induction of Leaf Tumors tered in response to infection with wild-type U. maydis; by con- After infection, U. maydis hyphae mainly grow intracellularly.
trast, only 773 transcripts were altered in response to infection About 4 d postinfection (DPI), small tumors are visible and the with SG200Δsee1 and fungus proliferates massively both intracellularly and intercellu- ). Hierarchical clustering of the SG200-induced maize genes larly. In mature tumors at 10 to 14 DPI, U. maydis forms masses visualized the reduced transcriptional response of maize leaves to of melanized teliospores (Doehlemann et al., 2008). Unlike other the see1 deletion mutant . A direct com- smut fungi of monocots, U. maydis causes these symptoms in parison of SG200 with SG200Δsee1 showed that 549 genes were

Tumor Induction by Ustilago maydis regulation of cell division (TC307447) increased 230-fold in wild-type infections . Together, these datasuggest that SG200Δsee1 fails to induce leaf tumor growth at thelevel of host cell DNA synthesis and cell proliferation, processesthat are hallmarks of maize responses to infection (Doehlemannet al., 2008).
See1 Is Required for U. maydis-Induced Plant DNASynthesis during Leaf Tumor Formation Because see1 expression is prominent during tumor enlargementand our initial observations indicated that SG200Δsee1 hyphaewere mainly restricted within mesophyll and bundle-sheath cells,we performed a more thorough confocal microscopy investigationof leaf infections. U. maydis-induced tumor growth reﬂects hostproliferation, then cell expansion; thus, DNA synthesis is a pre-requisite for growth. To monitor DNA synthesis in planta, wetreated uninfected and infected leaves with 5-ethynyl-2-deoxy-uridine (EdU) at several time points over a period of 5 h and thenharvested samples. Incorporation of EdU was visualized by at-taching a ﬂuorescent tag (AF-488). Maize nuclei were stained withpropidium iodide (PI) following a procedure described previouslyfor maize anthers (Kelliher and Walbot, 2011). In maize leaves at2 DPI, EdU treatment did not result in any detectable labeling. Weobserved this in maize leaves colonized with U. maydis and in Figure 1. Organ-Speciﬁc Phenotype of See1 Demonstrating Its Role in uninfected maize leaves (Figure 3A), suggesting that no or only rare, sporadic maize DNA synthesis occurs in seedling leaf blades (A) Disease symptoms caused by SG200Δsee1 in comparison with the during the early phase of infection. We conclude that in the in- wild-type progenitor strain SG200 in leaves and tassels. The mutant fected zones, the host cells were already postmitotic. By 4 DPI, shows a signiﬁcant reduction in leaf virulence. Maize seedling leaves were when the ﬁrst macroscopic symptoms appear in wild-type in- scored at 12 DPI. Disease symptoms in maize tassels were scored at 14 fections, EdU incorporation into leaf DNA was widespread (Figure DPI as described by Schilling et al. (2014). SG200, the virulent U. maydis 3A). Leaf cells invaded by fungal hyphae synthesized new DNA, progenitor strain; D, deletion mutant for see1; D/C, genetic complemen-tation of the deletion strain. The experiment was performed in three in- and this coincided with the induction of mitosis, which could be dependent biological replicates. n = number of plants infected. *P # 0.001.
visualized at different stages of cell division and as contiguous (B) Symptoms caused by U. maydis strain SG200 in comparison with the pairs of similarly labeled cells (Figure 3B). Such invaded cells also SG200Δsee1 mutant and the complemented strain in leaves and tassels.
underwent multiple division events over several days (Figure 3B).
The leaf photograph shows typical disease symptoms at 12 DPI; the As an additional negative control for the EdU labeling, we tassel photograph is at 14 DPI. Similar to SG200, the mutant caused injected 5 mM hydroxyurea, a DNA synthesis inhibitor, into disease symptoms in tassels, but leaf tumors were signiﬁcantly reduced.
seedling leaves infected with wild-type SG200 or SG200Dsee11 d before labeling them with EdU. Pretreatment with hydroxy- signiﬁcantly induced (>2-fold) in SG200 compared with SG200Δsee1 urea eliminated EdU incorporation in all samples; this was also at 6 DPI, while only two genes were repressed true in SG200-infected cells that already initiated division before The transcripts induced by infection with wild-type treatment. Hydroxyurea appears to block DNA synthesis com- pathogen were enriched for genes involved in DNA modiﬁcation pletely and validates the speciﬁcity of the EdU labeling assay (i.e., histones), DNA replication, and DNA damage repair as well Quantiﬁcation of EdU labeling showed as genes associated with the cell cycle ( that 67.5% 6 4.2% of the maize cells colonized by SG200 in- . Gene Ontology (GO) analysis showed that 71 of the 549 corporated EdU at 4 DPI (Figures 4A and 4C). Labeling was SG200-induced genes are associated with DNA metabolism and initiated from 3 to 4 DPI, while no DNA synthesis was observed cell cycle regulation (and in uninfected leaves of the same age. Therefore, U. maydis re- list the top 30 GO terms that are associated with DNA activated DNA synthesis and cell division in maize leaves at the metabolism and cell cycle regulation). As shown in Figure 2B, onset of tumor induction. By contrast, SG200Δsee1-infected DNA replicase D (TC280511), which is involved in S-phase DNA leaf samples showed only 7.3% 6 1.7% EdU-positive cells at replication, was induced 690-fold in wild-type infections com- 4 DPI (Figures 4A and 4C; The pared with SG200Δsee1. DNA histone H3 (TC298222), which is SG200Δsee1 deletion mutant fails to trigger DNA synthesis and required to generate nucleosomes, was induced 862-fold in wild- cell division to support the formation of large tumors.
type infections. Maize Skp1 (TC293032) was induced 875-fold in One might argue that the reduction in host cell DNA synthesis SG200 infections versus SG200Δsee1. Also, a Leu-rich repeat could be a general consequence of reduced virulence (i.e., receptor-like protein responsible for protein phosphorylation and an impaired biotrophic interaction); therefore, it would not be

observed (Figure 4A). This observation indicates that biotrophiccolonization of maize smut per se does not induce host DNAsynthesis. Reactivation of DNA synthesis is speciﬁc to tumorformation. Next, a U. maydis deletion mutant lacking the secretedeffector Tin3 was tested. Deletion of tin3 results in small leaf tu-mors similar to SG200Δsee1 strains (Brefort et al., 2014). Despite Figure 2. Gene Expression during Maize Colonization with SG200 andSG200Δsee1.
(A) RT-qPCR expression proﬁling of the see1 gene during the biotrophicphase of U. maydis growth in seedling and tassel tissues. Expressionlevels are shown relative to mean expression of ppi transcripts. Geneexpression was analyzed in axenic culture (AC), seedling, and tasseltissues at consecutive time points from 2 to 14 DPI. The experiment wasperformed in three independent biological replicates.
(B) Transcriptional regulation of the key genes involved in the process ofDNA synthesis and histone modiﬁcation between wild-type SG200- andSG200Dsee1 (mutant)-infected seedlings at 6 DPI. Hierarchical cluster-ing was performed by the Partek Genomics Suite version 6.12 to visu-alize the expression of maize genes transcriptionally regulated at 6 DPI Figure 3. U. maydis Induces DNA Synthesis in Infected Maize Seedlings.
by U. maydis strain SG200 (bottom), infection by SG200Dsee1 (middle),and mock inoculation (top). The x axis depicts clustering of the micro- (A) Maize seedlings were infected by U. maydis wild-type strain SG200, array samples for each of the three biological replicates for each treat- and then tissue was incubated in EdU to visualize in vivo DNA synthesis ment. The y axis shows clustering of the regulated maize transcripts in the host cells. Samples were imaged at 2 and 4 DPI by confocal mi- based on the similarity of their expression patterns. red, upregulated croscopy. Left, at 2 DPI, the fungal proliferation was observed sub- genes; green, downregulated genes; black, not signiﬁcantly altered. LRR, epidermally; host cells adjacent to fungal hyphae were considered to be Leu-rich repeat.
colonized cells (white arrowheads). No EdU incorporation was observed.
Right, at 4 DPI, numerous colonized cells showed EdU labeling (greenstain), indicating the onset of DNA synthesis in host cells (yellow ar- functionally linked with the action of See1. To test this, we in- rowheads). Bars = 75 mm.
cluded two additional strains. First, leaves were infected with wild- (B) Cell division events were observed in maize seedlings infected byU. maydis wild-type strain SG200 at 4 and 5 DPI. EdU incorporation into type strains of the maize head smut fungus Sporisorium reilianum, a cell will result in equally labeled contiguous daughter cells after cell a close relative of U. maydis. S. reilianum also establishes a bio- division. Such equally labeled cell pairs were readily observed in SG200- trophic interaction with maize, but it causes visible symptoms only infected seedling leaf tissue. The white arrowheads point to fungal hy- in the inﬂorescences, never in leaves (Schirawski et al., 2010).
phae associated with maize cells undergoing cell division. It is inferred Strikingly, at 4 DPI, the leaves infected with S. reilianum lacked that reactivation of the cell cycle and rapid divisions are responsible for detectable DNA synthesis, although dense tissue colonization was tumor formation. Bars = 25 mm.

Tumor Induction by Ustilago maydis Figure 4. See1 Requirement for Host Cell Cycle Release in Leaf Tumor Formation.
(A) In vivo DNA synthesis in seedling tissue infected with SG200Δsee1 in comparison with wild-type SG200. Samples infected with S. reilianum andSG200Δtin3, which has a similar phenotype to SG200Δsee1 with respect to tumor size, were used as controls. Fungal hyphae and plant cell walls werevisualized by PI staining (red), and the EdU-labeled host cell nuclei are visualized by AF488 staining (green). Fungal hyphae are shown by the whitearrowheads. Bar = 100 mm.
(B) DNA synthesis in anther tissue infected with SG200Δsee1 in comparison with wild-type SG200. Samples infected with the strain overexpressingSee1 and uninfected anthers served as controls (right panel). Nuclei were visualized by PI staining (red), and EdU-labeled host cell nuclei are visualizedby AF488 staining (green). Fungal hyphae are marked by white arrowheads. Bars = 100 mm.
(C) Quantiﬁcation of the EdU-labeled seedling leaf cells in the in vivo DNA synthesis assay comparing infections with wild-type SG200, SG200Δsee1,SG200Δtin3, and S. reilianum. Error bars show SE. *P # 0.001.
(D) Quantiﬁcation of the EdU-labeled nuclei relative to total anther nuclei per image examined after infection with wild-type SG200, SG200Δsee1, See1-overexpressing strain Ppit2-see1, and noninfected (N.I) tissue. Within the population of EdU-positive cells, the number colonized by fungal hyphae wasalso quantiﬁed in the infected samples. Error bars show SE.
its severely reduced virulence, the SG200Δtin3 deletion mutant indirect consequence of reduced tumor size but reﬂects a required activated EdU labeling in 44.22% 6 4.0% of colonized leaf cells at activity of the See1 effector.
4 DPI (Figures 4A and 4C). Therefore, there is more than onecause of impaired tumor induction, separating See1 from other Tumor Formation in Anthers Does Not Involve mutants that lack large tumors but retain the ability to reactivate U. maydis-Induced DNA Synthesis widespread host DNA synthesis. From these results, we concludethat the inability of the SG200Δsee1 mutant to reactivate maize The reproductive spikelets each contain two ﬂorets with three an- cell DNA synthesis and proliferation for tumor formation is not an thers and arise within the tassel inﬂorescence; the nonreproductive ﬂoral tissues such as the glumes, palea, and lemma of each spikelet, with SG200, SG200Δsee1, and noninfected samples (Figures 4B as well as the tassel stem, are readily infected and transformed to and 4D), further evidence that See1 is not involved in tumor for- tumors by U. maydis. Interestingly, the fungus is only effective in mation in anthers. In the terminal node/internodes, however, the causing anther tumors during the period of rapid anther growth by frequency of EdU-labeled cells was signiﬁcantly increased by see1 cell division prior to meiosis (Walbot and Skibbe, 2010). The intrinsic overexpression as compared with SG200 (Ppit2-see1, 31.8% 6 anther developmental program of rapid proliferation is reprogram- 4.5%; SG200, 20.7% 6 4.5%; mock, 8.1% 6 1.6%). Therefore, med into a tumor pathway, with different cell types affected de- the abnormal phenotype is a direct consequence of the excessive pending on when fungal hyphae invade the cells (Gao et al., 2013).
cell division resulting from see1 overexpression (Figures 5C and From this observation, Gao et al. (2013) concluded that tumor for- 5D). In summary, we conclude that See1 is required to stimulate mation in anthers mainly occurs by restructuring of the usual se- U. maydis-induced tumor formation by promoting host DNA quential events in cell fate speciﬁcation. In line with this hypothesis, synthesis in vegetative tissue but not in maize anthers.
we did not observe signiﬁcant differences in EdU-labeled cells inuninfected tissue compared with U. maydis-infected anther tissue.
See1 Localizes to Maize Cytoplasm and Nuclei Uninfected anthers as well as SG200- and SG200Δsee1-infectedpremeiotic anthers contained ;60% cells labeled with EdU in a 5-h Live cell imaging and immunolabeling using transmission elec- treatment (Figures 4B and 4D). This is consistent with the previous tron microscopy (TEM) were used to localize See1 in planta. An report that, during the rapid proliferation period of anthers, EdU in- mCherry-tagged version of See1 lacking its N-terminal secretion corporation was found in the majority of cells after a 4-h labeling signal (35S promoter:See1 -mCherry) was transiently expres- (Kelliher and Walbot, 2011). Therefore, in contrast with leaves, sed in maize leaves by particle bombardment (Figure 6A).
U. maydis does not alter anther cell DNA synthesis. In line with -mCherry localized to both the maize cytoplasm and this, ;42% of the EdU-positive anther cells were colonized by U.
nuclei (Figure 6A). As a transformation control, the nuclear marker maydis at 4 DPI, with no signiﬁcant difference between SG200 and protein PCNA-interacting protein (PIP )-yellow ﬂuorescent SG200Δsee1. We conclude that See1 is not involved in modulating protein (YFP) was coexpressed and localized exclusively to the host DNA synthesis and cell division during colonization and tumor nuclei. As a localization control, mCherry expressed alone and induction in anthers and that it is dispensable for tumor formation in Pit2-mCherry (Pit2 -mCherry) also showed the same locali- zation pattern to the cytoplasm and nucleus when transientlyexpressed in maize epidermal cells ). Interestingly, the See1 -mCherry signal spread to See1 Actively Contributes to Tumor Formation and Maize the nuclei of cells surrounding individual transformed cells (Figure 6A; ). Fluorescence signal movement To test whether See1 actively contributes to tumor formation was not observed for mCherry or for the Pit2-mCherry fusion and DNA synthesis, a U. maydis strain was generated that con- protein (. These results in- stitutively expresses see1 during the entire infection process, in- dicate that See1 may trafﬁc in planta.
dependently of the colonized tissue. See1 was expressed from the In addition to the heterologous expression of See1 in maize promoter of pit2, which is one of the U. maydis genes with the cells, the effector was localized upon natural delivery (i.e., when strongest in planta transcription (Skibbe et al., 2010; Doehlemann it was secreted from infectious U. maydis hyphae). A C-terminal et al., 2011). In leaf infections, the Ppit2-driven overexpression of 3xHA-tagged version of the effector was expressed under the see1 RNA (veriﬁed by reverse transcription and quantitative PCR control of the native promoter in the SG200Δsee1 deletion strain.
[RT-qPCR]) and protein (conﬁrmed by immunoblot analysis) did Immunoblot analysis after immunoprecipitation of See1-3xHA not result in a phenotype signiﬁcantly different from the wild type from infected plant tissue veriﬁed the expression and stability of Therefore, ectopic overexpression the fusion protein ). For the immunoloc- of see1 did not augment the virulence potential of U. maydis in alization of See1-3xHA, plants infected with U. maydis strain leaves. Interestingly, the see1-overexpressing strain caused an SG200 served as negative controls. Two additional controls for unexpected tassel phenotype, although tassel tumors caused immunolabeling were employed: strain SG200 Psee1-GFP-3xHA by wild-type U. maydis are largely restricted to the ﬂoral tis- expresses cytoplasmic green ﬂuorescent protein (GFP) driven by sues, particularly the anthers. By contrast, Ppit2-driven see1 the see1 promoter, and strain SG200 Psee1-SPsee1-mCherry- expression caused extensive tumor formation in the vegetative 3xHA expresses a secreted mCherry under the control of the tassel base (the terminal node and internode) (Figure 5A). This see1 promoter. Maize leaves were inoculated, and samples were effect, which resulted in bizarre alterations of tassel architec- harvested at 6 DPI for immunogold detection of the 3xHA tag. In ture, was observed in ;38% of the infected plants (wild type, the TEM images, no gold labeling was seen in plant tissue in- 8%) Additionally, ;20% of fected with the parental strain SG200, indicating the absence infected tassels including the spikelets became green 10 DPI of nonspeciﬁc background labeling (Figure 6B, left panel). The with the see1-overexpressing strain ( nonsecreted GFP-3xHA was detected exclusively inside fungal hyphae at established biotrophic interfaces (Figure 6B, middle Tissue infected by the see1-overexpressing strain was also panel). The secreted mCherry-3xHA control showed labeling used to quantify EdU-labeled cells in anthers as well as the mainly in the biotrophic zone surrounding fungal cells as vegetative tassel base. In anthers, overexpression of see1 did well as inside fungal hyphae (Figure 6B, right panel). By not cause any signiﬁcant differences in EdU labeling compared contrast, See1-3xHA was detected in the fungal hyphae, at the

Tumor Induction by Ustilago maydis Figure 5. Overexpression of see1 Results in Tumor Proliferation in Vegetative Tassel Parts.
(A) Tassel base abnormality occurs much more frequently with constitutive overexpression of see1 in comparison with the wild-type strain SG200.
Tumor formation in the tassel base is indicated by the white arrows.
(B) Quantiﬁcation of see1 gene expression in tassels infected with the overexpressing strain Ppit2-see1 in comparison with plants infected with the wild-type SG200 strain. Error bars show SE. *P # 0.001.
(C) Quantiﬁcation of EdU-labeled tassel base cells in the in vivo DNA synthesis assay after infection with the See1-overexpressing strain Ppit2-see1 incomparison with either the wild-type SG200 infected or noninfected (N.I) tassels. There was a signiﬁcant difference in the number of EdU-labeled nuclei inthe abnormal tassel base region as compared with the wild-type SG200 infected or noninfected tissue. Error bars show SE. Comparisons a and b, P # 0.05.
(D) Detection of in vivo DNA synthesis in the tassel base colonized by See1-overexpressing strain Ppit2-see1 in comparison with tissue colonized bywild-type strain SG200 and noninfected tissue. The total nuclei were visualized by PI staining (red), and the EdU-labeled cell nuclei were visualized byAF488 staining (green). Bar = 50 mm.
biotrophic interface, in plant cytoplasm, and prominently inside found in the biotrophic interface. Only See1-3xHA was quan- plant cell nuclei (Figure 6C; The dis- titatively detected inside host cells, with ;20% of particles tribution of gold particles was quantiﬁed for all constructs.
localizing to maize nuclei (Figure 6D). These results conclu- While all the encoded proteins were found inside fungal cells, sively demonstrate the translocation of See1 from biotrophic only the two proteins with N-terminal secretion signals were fungal hyphae into maize cytoplasm and nuclei.

Figure 6. See1 Localizes to the Plant Cell Cytoplasm and Nucleus.
(A) Confocal microscopy of 35S-see1 -mCherry transiently expressed in maize epidermal cells. Left panel, transformation with the PIP-YFP control results in ﬂuorescence that is speciﬁcally localized to the nucleus. Right panel, See1-mCherry is localized to the cytoplasm and nucleus and istransferred to the adjacent neighboring cells, which are shown by the white arrowheads in the mCherry and overlay channels. Bar = 25 mm.
(B) Controls for the TEM micrographs showing immunogold labeling of See1-3xHA in leaves of U. maydis-infected maize. No gold particles were boundto wild-type infected tissue specimens (left panel). Gold labeling was restricted to fungal hyphae in GFP-3xHA samples, as GFP was not secreted by thefungus (middle panel). Gold particles bound to the secreted mCherry control could be found in hyphae and at the biotrophic interface (red arrowheads)but not inside the plant cells, despite proximity to hyphae. Psee1-SPsee1-mCherry-3xHA expression demonstrates that mCherry is secreted by thefungus but not taken up by the plant (right panel). Bars = 1 mm.
(C) Immunogold labeling of See1 could be found in hyphae (H), the cytosol, and nuclei (N), as shown by the red arrowheads, but not in chloroplasts (C),vacuoles (V), or the cell wall (CW) when the See1 effector was tagged with 3xHA in the strain Psee1-SPsee1-See1-3xHA. Bar = 1 mm.
(D) Graph depicts the spatial distribution of gold particles bound to See1-3xHA in different cell compartments of leaves from Psee1-See1-3x-HA alongwith the secretory (mCherry-3xHA), nonsecreted (GFP-3xHA), and SG200 wild-type controls. Means are shown with SE for the number of gold particlesper mm2 in the individual cell compartments of three independent transverse sections. Lowercase letters indicate signiﬁcant differences (P < 0.05)between the individual cell compartments, whereas uppercase letters indicate signiﬁcant differences between the total sum of labeling signal for allanalyzed cell compartments. Data were analyzed with the Kruskal-Wallis test followed by post-hoc comparison according to Conover (1999). n.d., notdetected, for all analyzed cell compartments.
Tumor Induction by Ustilago maydis See1 Interacts with a Maize Homolog of SGT1 See1 Interferes with the Phosphorylation of SGT1 To identify proteins interacting with See1, we performed a yeast A major question arising from the See1 interaction with SGT1 two-hybrid screen using a normalized cDNA library of U.
concerns how the effector interferes with SGT1 function at the maydis-infected maize leaves and tassels. From 60 clones that molecular level. Recent work showed that activation of SGT1 were isolated after plating on high-stringency selection medium, signaling activity requires the phosphorylation of SGT1 by sali- sequences corresponding to a maize homolog of SGT1 were cylic acid-induced protein kinase (SIPK), a MAPK activated in identiﬁed (Figures 7A and 7B). SGT1, a known regulator of cell response to pathogen assault (Hoser et al., 2013). SIPK-mediated cycle progression in yeast and an important factor in plant host phosphorylation of SGT1 was concluded to trigger enhanced and nonhost resistance, has three functional domains: the tetra- nuclear compartmentalization of SGT1, thereby possibly activat- tricopeptide repeat (TPR) domain, the Chord SGT1 (CS) domain, ing defense-related signaling via the modulation of transcription.
and an SGT1-speciﬁc (SGS) domain (Figure 7A) (Kitagawa et al., Based on these observations, we tested whether See1 interferes 1999; Peart et al., 2002). There are also two variable protein re- with the phosphorylation of SGT1. To facilitate the analysis of the gions that are species-speciﬁc (Figure 7A). To test whether the in planta phosphorylation of SGT1 in the presence of See1, we identiﬁed maize protein exhibits SGT1 functions, temperature- used the well-established Agrobacterium tumefaciens expression sensitive mutants of S. cerevisiae, YKK57 (sgt1-5) and YKK65 system in N. benthamiana. N. benthamiana SGT1, which shares (sgt1-3) (Kitagawa et al., 1999), were used for complementation 65% identity to maize SGT1 ), was pre- experiments. At 37°C, both mutants are restricted in cell cycle viously shown to be activated by Nt-SIPK, which, in turn, shares phases, arrested at G1 and G2, respectively. Expression of full- 84% to 86% identity to a set of ﬁve putative MAPK proteins of length maize SGT1 under the control of the GAL4 yeast promoter maize ). Agrobacterium strains carrying complemented the growth defect of S. cerevisiae strain YKK57 P35S-Zm-SGT1-StrepII, a gene encoding constitutively active (sgt1-5), indicating the functionality of the identiﬁed maize ho- Nt-MEK2 (Nt-MEK2DD) under the control of a dexamethasone molog. Expression of maize SGT1 in S. cerevisiae strain YKK65 (DEX)-inducible promoter, P35S-SIPK, and Dex-see1-HA or pTA7001 (sgt1-3), which is defective at G2, showed normal growth at empty constructs were coinﬁltrated into N. benthamiana leaves permissive temperature and partial complementation at 37°C and maintained for 2 d to ensure expression of the constitutive promoter constructs. Subsequently, Nt-MEK2DD and See1 ex- To verify the See1-SGT1 interaction in planta, both proteins pression were induced by DEX treatment. Leaf samples were were transiently expressed in Nicotiana benthamiana. As expression collected 5 h after the induction, and SGT1 was afﬁnity-puriﬁed controls, P35S-See1-Myc and P35S-SGT1-HA were separately via its Strep tag II. Expression of all heterologous proteins has expressed in N. benthamiana leaves (Figure 7C). Using anti-HA been veriﬁed by immune detection We matrix, See1-Myc was coimmunoprecipitated by the HA-tagged detected two sites of phosphorylation in Zm-SGT1. Constitutive SGT1 but not in the absence of SGT1 (Figure 7C), conﬁrming phosphorylation that was independent from See1 as well as the See1-SGT1 interaction in planta. To localize the See1- from SIPK was detected for residue Thr-262 SGT1 interaction in plant cells, bimolecular ﬂuorescence and This residue is situated in complementation (BiFC) was employed, using an enhanced the second variable region of Zm-SGT1 and only conserved in split-YFP system (Hemetsberger et al., 2012). An mCherry tag maize and sorghum (Sorghum bicolor) was fused to the C terminus of the N-terminal part of YFP Surprisingly, upon SIPK activation and in the absence of See1, (pSPYNE_N). Similarly, a cyan ﬂuorescent protein (CFP) tag there was a high abundance of a Zm-SGT1 phosphopeptide was added to the C-terminal part of YFP (pSPYCE_C). Via with phosphorylation at Thr-150 (Figures 8A and 8B) in all three ballistic transformation of maize epidermal cells, both con- independently performed biological replicates. This phosphor- structs were transiently expressed under the control of the 35S ylated position is situated in the variable region of Zm-SGT1, promoter. Cells expressing both pSPYCE_C and pSPYNE_N represents a putative MAPK target site, and is conserved in fused to SGT1 and See1, respectively, were designated as monocots including maize, rice (Oryza sativa), and sorghum ). Strikingly, in See1 coinﬁltrated samples, CFP-CYFP-HA. The cells exhibited cytoplasmic and nuclear no phosphorylation at Thr-150 could be detected, reﬂecting the ﬂuorescence signals for both mCherry and CFP, indicating interference of See1 with SIPK1-induced phosphorylation of SGT1 expression of the fusion proteins. Expression of pSPYNE_N- and ). As a negative mCherry with pSPYCE_SGT1 did not result in any detectable control, we coexpressed an inactive form of Nt-MEK2 (Nt-MEK2KR) YFP signal, demonstrating that no unspeciﬁc protein di- with SGT1. In this case, SIPK was not included because its tran- merization occurred (Figure 7D). Similarly, no YFP ﬂuorescence sient expression results in slightly increased SIPK activity and the was detected when pSPYCE-CFP was coexpressed with associated activation of defense responses (Zhang and Liu, 2001).
pSPYNE-mCherry fused to see1 (pSPYNE_see1) (Figure 7D). By Under these conditions, coexpression of Nt-MEK2KR and SGT1 did contrast, cells that coexpressed pSPYNE_see1 and pSPYCE_SGT1 not result in SGT1 phosphorylation at Thr-150. Therefore, we showed a complementation of YFP ﬂuorescence (Figure 7D), conclude that See1 interferes with the Zm-SGT1 phosphorylation at indicating an interaction of See1 and SGT1 in the cytoplasm Thr-150, thereby delaying or preventing its activity.
and nucleus of maize cells. Altogether, we demonstrate that To corroborate the result that SIPK could phosphorylate Zm- See1 interacts with SGT1 in the cytoplasm and nucleus of SGT1, puriﬁed recombinant proteins Zm-SGT1, SIPK, and MEK2DD maize cells.
were coincubated in the presence of radioactive ATP in vitro. As

Figure 7. See1 Interacts with the Cell Cycle and the Immune Response Modulator SGT1.
(A) Domain structure of maize SGT1 depicting three important domains: TPR, CS, and SGS. The two variable regions (VR1 and VR2) in the protein arespecies-speciﬁc.
(B) Yeast two-hybrid experiment to test for the interaction of See1 and maize SGT1. The drop assay was done by serial dilutions (see Methods), andstrains were tested on low- and high-stringency plates to check for the speciﬁcity of the interaction. Results were documented after 4 d.
(C) Coimmunoprecipitation shows the interaction of See1 and SGT1 fusion proteins isolated from transiently expressing N. benthamiana cells. SGT1was tag puriﬁed, and See1 was pulled down. In the absence of SGT1, no See1 signal was detected.
(D) In vivo interaction of See1 with SGT1. Confocal images show maize epidermal cells expressing BiFC constructs. Row I shows a plant cellcoexpressing pSPYCE-SGT1 and pSPYNE-mCherry. Blue and red channels show cytoplasmic colocalization of the respective signals. No comple-mentation of ﬂuorescence is observed in the YFP channel. Row II shows the coexpression of pSPYCE-CFP and pSPYNE-See1. Blue and red channelsshow cytoplasmic colocalization of the respective signals. No complementation of ﬂuorescence is observed in the YFP channel. Row III shows a cellcoexpressing pSPYCE-SGT1 and pSPYNE-See1. Both signals colocalize in the nucleus and cytoplasm. The YFP channel exhibits YFP ﬂuorescencereﬂecting the direct interaction of See1 and SGT1. DIC, differential interference contrast. Bars = 25 mm.
shown in the SIPK activated by a positive control, a nonspeciﬁc substrate, myelin basic protein MEK2DD was able to phosphorylate Zm-SGT1 (lane 1). In the lane 3), was used; this protein was in- negative control, which did not contain SIPK, we did not observe tensively phosphorylated in our assay. Collectively, from these any phosphorylation of Zm-SGT1, and the signal detected cor- results, we conclude that SIPK from tobacco (Nicotiana tabacum) responded to MEK2DD (, lane 2). As can phosphorylate Zm-SGT1 in planta or in vitro and, therefore,

Tumor Induction by Ustilago maydis Figure 8. In Planta Phosphorylation of Maize SGT1.
(A) Fragmentation spectrum assigned to the phosphorylated form of the peptide EDVANMDNTPPVVEPPSKPK (Mascot score 126). Loss of H PO is denoted by –P, loss of water is marked by short horizontal arrows, whereas a longer arrow symbolizes pairs of detected signals corresponding to y and y -H PO . The majority of signals of the tandem mass spectrometry spectra are assigned to the above species. The presence of several y and b -H PO ions accompanied by y , y , and y pinpoints threonine at position 9 as the unequivocal phosphorylation site within the peptide.
(B) Peptide sequence with assigned y, b, y-H O, b-H O, y-H PO , and b-H PO ions present.
(C) Recombinant maize SGT1 produced in E. coli was incubated in the buffer containing [g-32P]ATP, and total proteins were extracted from maize seedlingsor tassels infected by various U. maydis strains. The samples were fractionated by SDS-PAGE and analyzed with a phosphor imager. Columns 1 to 3,extracts from seedling leaves 6 DPI with U. maydis wild-type SG200 (1), SG200Δsee1 (2), or mock-inoculated (3). Columns 4 to 6, extracts from tassel base9 DPI with U. maydis wild-type SG200 (4), U. maydis-overexpressing Ppit2:see1 (single-copy integration; 5), or U. maydis-overexpressing Ppit2:see1(multiple-copy integration; 6). Representative data of four independent biological replicates are shown. CBB, Coomassie Brilliant Blue.
could be used for transient assays to study the See1-SGT1 while a phosphomimic variant of At-SGT1b exhibited enhanced nuclear localization (Hoser et al., 2013). To test whether the Phosphorylation can alter protein subcellular localization and phosphorylation of Zm-SGT1 altered its subcellular localization, stability (Cohen, 2000). A truncated Arabidopsis thaliana SGT1b constructs encoding YFP fused C-terminally to the wild-type lacking a MAPK phosphorylation site was detected only in the Zm-SGT1 or to its phosphovariants were transiently expressed nucleus-depleted fraction in leaf extracts (Noël et al., 2007), in N. benthamiana epidermal cells via particle bombardment.
Wild-type Zm-SGT1 localized to the nucleus in ;33% of the constructs), tassel bases showed quantitative reduction of SGT1 transformed cells, and its relative nuclear fraction in all cells phosphorylation (Figure 8C). When using Zm-SGT1 protein with tested was 39.4% Upon bombard- the T150A (SGT1AP) mutation in the same assay, residual phos- ment with constructs encoding Zm-SGT1 fused to a strong phorylation was observed mainly in the tassel tissue nuclear localization signal derived from SV40 large T antigen or . This signal did not correlate with See1 expression and, protein serine kinase protein-derived nuclear export signal, the therefore, most likely represents the previously observed consti- nuclear fraction was changed to 94.3% and 15% of the trans- tutive phosphorylation of residue Thr-262 ( formed cells, respectively (Wen et al., 1995). Control-like subcellular and Together, these ﬁndings demon- distribution could be restored by mutation of either the nuclear lo- strate that Thr-150 phosphorylation of maize SGT1 is triggered in calization signal or the nuclear export signal response to U. maydis infection and that this is modulated by the , demonstrating the suitability of the bombardment assay to See1 effector.
assess protein localization. The phosphonull (Zm-SGT1AP) variantlocalized to the nucleus in around 20% of the transformed cells, and the nuclear fraction was 33.5% . Bycontrast, the phosphomimic variant of Zm-SGT1 (Zm-SGT1DP) The secreted effector protein See1 is an organ-speciﬁc U. maydis was observed in the nucleus of ;40% of transformed cells, with virulence factor that promotes tumor formation in maize vegeta- an average nuclear fraction of 46.2%. These data suggest that tive tissues. The expression proﬁle of see1 shows strong organ Zm-SGT1 phosphorylation status affects nuclear import or export.
speciﬁcity, supporting the speciﬁc requirement for this effector in Because the differences are subtle compared with the wild type, we supporting tumors in maize leaves but not in ﬂoral tissues. See1 is conclude that the disturbance in localization of the Zm-SGT1 is not translocated from biotrophic hyphae to the maize cell cytoplasm the primary virulence function of See1.
and nucleus, where it interacts with maize SGT1 and interfereswith its MAPK-induced phosphorylation. In leaf zones with post-mitotic differentiated cells, U. maydis requires See1 to reactivate The See1-SGT1 Interaction Has Functional Relevance host cell division, a prerequisite for tumor formation. By contrast, To further investigate the role of See1 in targeting SGT1, we anther tumor induction does not require de novo activation of checked if the identiﬁed phosphorylation at Thr-150 in Zm-SGT1 plant cell proliferation, because in this highly proliferating tissue, is required for SGT1 function. To address this point, we ﬁrst the tumors result from redirecting host cell division and cell ex- performed a yeast complementation assay using sgt1 cell cycle pansion into a tumor pathway (Gao et al., 2013).
mutants to test for the complementation ability of phosphomi- During maize leaf development, most cell divisions occur in mic and phosphonull mutants of Zm-SGT1 at this position. This a narrow zone at the base of the blade adjacent to the ligule, assay did not show any differences in the complementation with only sporadic divisions in the differentiating leaves. Tumors ability compared with the wild-type SGT1 formed in U. maydis infections result from the profuse and rapid This suggests that the phosphorylation site we identiﬁed in cell division in the subepidermal leaf cells. In uninfected plants, planta is not required for the cell cycle-related SGT1 function in no such activity was visible in the corresponding leaf area (which yeast. Because the relevant phosphorylation site is speciﬁc to is neither part of the apical meristem nor the basal region of the monocot SGT1 homologs and not present in yeast, one could leaf) (Li et al., 2010). Confocal microscopy of leaves infected by not expect a function of this residue in yeast.
SG200Δsee1 showed that it successfully penetrated the host To elucidate the relevance of SGT1 phosphorylation in tumor tissue and established itself in the initial stages of colonization.
formation, an alternative approach was employed. Speciﬁcally, During the later stages, when the fungi reached the mesophyll this aimed to identify the phosphorylation status of SGT1 upon cell layer, the mutant displayed defects in passing from cell to U. maydis infection and to further test the phosphorylation state cell, with entrapment of the fungal hyphae in the mesophyll cells in overexpression strains as compared with the U. maydis wild- or the adjacent vascular cells This stage type SG200. To this end, maize leaves were infected with the of infection at 4 DPI coincides with the normal appearance of wild-type U. maydis strain SG200 and SG200Dsee1. In addition, heavy EdU labeling, indicative of reinitiation of the cell cycle in tassels were infected with strains overexpressing See1 (which leaves infected with the wild-type fungi, as monitored by DNA caused the abnormal tassel base phenotype) to collect the vege- precursor incorporation (Gratzner, 1982; Salic and Mitchison, tative tassel base tumors. An in vivo detection of SGT1 phos- 2008). EdU labeling of seedling tissue colonized by U. maydis phorylation was performed by preparing extracts from infected showed that several division events had occurred in contiguous tissues, which served as a kinase source in the assay. Supple- cells by 4 to 5 DPI, indicating that reactivation of host cell division mental recombinant Zm-SGT1 in maize leaf or tassel base extracts occurs after initial fungal establishment and is followed by sus- was monitored for its phosphorylation state by autoradiography. It tained proliferation of maize leaf cells.
is clear that Zm-SGT1 is phosphorylated after incubation with the During the initial 2 d of anther colonization, U. maydis is present extracts from U. maydis-infected leaves as compared with the on the epidermis (Gao et al., 2013). At later time points, the fungus uninfected control (Figure 8C). Moreover, incubation with the ex- is subepidermal and alters cell fate speciﬁcation events, ongoing tracts from leaves infected with SG200Dsee1 showed increased cell division patterns, and cell expansion depending upon de- phosphorylation of SGT1 compared with the extracts from wild- velopmental stage and cell type. The fungus mainly induces type SG200-infected tissue. With U. maydis overexpression of ectopic periclinal divisions in anther somatic cells, generating See1 (by single and multiple integration of overexpression an extra cell layer resulting in disrupted anther lobe architecture.
Tumor Induction by Ustilago maydis Frequent anticlinal and periclinal divisions are also observed in subsequent cell-to-cell movement may reﬂect a general feature of the middle layers of infected anther, which otherwise undergo effectors taken up by plant cells. These proteins could act to only a few anticlinal divisions after their birth, prior to programmed stimulate the surrounding cells not yet in contact with fungal hy- cell death. Hence, in ﬂoral tissues, U. maydis reprograms cell fate phae to promote fungal proliferation. For See1, the independent but does not act as an oncogenic agent (Gao et al., 2013).
approach of immunolabeling clearly conﬁrmed that the effector is Constitutive overexpression of see1 led to tumor formation in the translocated to the plant cell cytoplasm and nucleus. Movement vegetative parts of the tassel base, which in wild-type infections of See1 to the neighboring cells was not observed in the TEM were not tumorous under the tested conditions. This suggests immunogold assay. This discrepancy might result from different that See1 speciﬁcally acts in vegetative tissues. The phenomenon expression levels in the two approaches; in the immunolabeling, might be of signiﬁcance to the fungus in nature, where ﬂoral tu- See1 was expressed by fungal hyphae from its native promoter, mors are more frequent in occurrence. See1 and other leaf- but plant-derived expression was driven by the 35S promoter.
speciﬁc effectors might be of importance for host adaptation and Nevertheless, based on our data, we cannot exclude that the cell- the evolution of U. maydis, as they promote the formation of tu- to-cell movement observed in the transient assay is an experi- mors in vegetative parts, an important factor in colonizing pe- mental artifact of the exogenous expression of the effector rather rennial grasses or exploiting seedlings. Infection of seedlings and than a feature of normal infections. On the other hand, the phe- immature plants allows the fungus to complete its short, 2-week nomenon of cell-to-cell movement of translocated effectors has life cycle multiple times during plant vegetative growth, because also been described for Magnaporthe oryzae effectors in rice cells this infection style is independent from the development of plant (Khang et al., 2010). This movement between cells was proposed to occur via the plasmodesmata, which are coopted by hyphae for Where does the See1 effector act in leaf cells? Transient ex- cell-to-cell movement (Kankanala et al., 2007; Djamei et al., 2011).
pression assays showed that See1 localizes to the cytoplasm See1 interacts with maize SGT1 in a yeast two-hybrid screen.
and nucleus of maize cells. Interestingly, the effector protein Maize SGT1 is present in both the cytoplasm and nucleus of also moved to cells neighboring a transformed cell. The speciﬁc plant cells; hence, localization is consistent with the dual locali- translocation of See1-mCherry between neighboring maize cells zation of See1. The interaction of See1 and SGT1 was conﬁrmed suggests that there may be a second route for fungal effectors independently by in planta coimmunoprecipitation. In addition, to enter maize cells. The U. maydis effectors Cmu1 and Tin2 BiFC data showed that this interaction takes place in both the are both translocated from biotrophic hyphae into the host cytoplasm and nucleus of maize cells. This evidence is congruent cell (Djamei et al., 2011; Tanaka et al., 2014). We speculate that with the electron microscopy analyses that show the same Figure 9. Tentative Model of the Role of the See1-SGT1 Interaction during U. maydis Tumor Formation.
The SGT1 protein is known to occur in the cytoplasmic and nuclear pools (Hoser et al., 2013). In U. maydis wild-type infections, activated unidentiﬁedmaize kinase (UMK) triggers the phosphorylation of SGT1 at a monocot-speciﬁc target site. The See1 effector binds to SGT1, interferes with itsphosphorylation status, and thereby disturbs the subcellular distribution (i.e., transport into the nucleus). This misbalancing of SGT1 phosphorylationand distribution contributes to the induction of cell cycle genes, leading to the induction of tumorous division.
localization pattern of the See1 effector. Mass spectrometry for symptom development. The precise steps following the in- analysis showed that the interaction with See1 results in an in- terference of See1 with the posttranslational modiﬁcation of hibition of SGT1 MAPK-triggered phosphorylation at Thr-150.
SGT1, resulting in the reactivation of maize DNA synthesis and Why would See1 interfere with the phosphorylation of SGT1 ultimately in tumor formation, remain to be elucidated biochemi- during leaf infection? Hijacking of SGT1 may contribute to the cally. Therefore, it will be of prime interest to work out the detailed deactivation of immune responses. The literature provides a large molecular mechanism of the See1-SGT1 interaction and to dissect body of data on SGT1. From all the available evidence concerning the downstream signaling network (i.e., to identify and characterize plant SGT1, this protein is widely seen to be active in vegetative proteins that interact with and/or are affected by SGT1 and to leaf tissues (Noël et al., 2007). In Arabidopsis, the SGT1b isoform pinpoint residues in See1 required to interfere with SGT1).
was found to be required for the SCF-mediated auxin response in Transcriptome analysis of U. maydis wild-type and SG200Δsee1- seedling roots (Gray et al., 2003). Arabidopsis SGT1 has a regu- infected maize leaves at 6 DPI showed that genes involved in latory role in early R gene-mediated plant defenses (Austin et al., DNA binding, replication (including Skp1), as well as repair mech- 2002) and was shown to be involved in forming a cochaperone anisms characterize normal infections but are not induced in the complex with HSP90; this complex functions in sensing immune mutant interaction. We hypothesize that this reﬂects the reduced responses of the host receptor proteins (Shirasu, 2009). Among activation of cell division and, consequently, limited tumor expan- the three domains of SGT1, namely the TPR, CS, and SGS do- sion in the mutant-infected leaves. Recent work by Bao et al. (2013) mains, the CS domain resembles the a-crystalline domain of the found an unexpected link between cell cycle progression and plant cochaperone HSP20 (Dubacq et al., 2002; Garcia-Ranea et al., immunity, suggesting that cell cycle misregulation impacts the ex- 2002). The other components of the immune regulatory cocha- pression of R genes. Also, DNA repair proteins have been shown to perone complex, RAR1 and HSP90, have been shown to interact be directly involved in the regulation of gene expression during with the CS domain of SGT1 (Azevedo et al., 2002; Takahashi plant defense responses (Song et al., 2011). The DNA damage et al., 2003).
response is an intrinsic component of the plant immune response The host SGT1/RAR1/HSP90 complex is a target of several and, in turn, enhances salicylic acid-mediated defense gene ex- bacterial effector proteins. The Pseudomonas syringae effector pression (Yan et al., 2013). Among the Ustilaginales, which cause AvrB interacts weakly with Arabidopsis SGT1b (Cui et al., 2010).
characteristic ﬂoral symptoms in the immature, proliferative host Another P. syringae effector, AvrPtoB, showed a genetic in- ﬂoral organs, U. maydis is the only species that causes local tumors teraction with SGT1 and RAR1, requiring these cochaperones to in vegetative tissue. Seedling-speciﬁc effectors such as See1 pro- suppress plant immunity (Hann and Rathjen, 2007). Additionally, mote the generation of a mitotically active sink tissue within vege- the P. syringae effector HopI1 interacts with HSP70 (Jelenska tative organs. By regulating SGT1, See1 may not only shut down et al., 2010), which is an active component initiating signaling by defense signaling but also activate the host cell cycle, a prerequisite interaction with the SGT1/RAR1 complex. Recently, effector for tumor development. Hence, a combination of immune sup- proteins from Salmonella enterica and Xanthomonas campestris pression and nutrient rechanneling, particularly facilitating the ac- have been shown to interact with SGT1 (Bhavsar et al., 2013; quisition of sucrose, could trigger uncontrolled cell proliferation, Kim et al., 2014). Consistent with the previous ﬁndings, these ultimately resulting in plant tumors. Based on the results obtained in effectors bind to SGT1 at the CS domain, conﬁrming the impor- this study, our hypothesis on how the See1-SGT1 interaction could tance of this domain for SGT1 regulation during immune de- affect the formation of leaf tumors is shown in a tentative model fenses. SGT1-mediated pathways may vary by plant species and (Figure 9). Detailed understanding of such processes should shed are also speciﬁc to a particular pathogen (Wang et al., 2010).
light on colonization biology in various biotrophic plant pathogens SGT1 is known to be used by some fungal pathogens in pro- and on intrinsic host mechanisms that operate to prevent cell moting disease symptoms. Like X. campestris, the necrotrophic proliferation in differentiated organs.
fungus Botrytis cinerea uses SGT1 to initiate the hypersensitiveresponse-mediated cell death pathway for the necrotrophic life-style (El Oirdi and Bouarab, 2007). Fusarium culmorum is known to require SGT1b to cause full disease symptoms in buds andﬂowers of Arabidopsis (Cuzick et al., 2009), the only prior report Growth Conditions and Virulence Assays that described SGT1 in ﬂoral tissues. These observations suggest Maize (Zea mays cv Early Golden Bantam [Olds Seeds] and cv Gaspe Flint that searching for a tissue-speciﬁc role of SGT1 protein in Ara- [maintained by self-pollination]) plants were grown in a temperature- bidopsis ﬂowers may be fruitful.
controlled greenhouse (14-h/10-h light/dark cycle, 28/20°C). Gaspe Flint We propose that SGT1 represents a conserved hub targeted plants were mainly used for tassel infection and for the overexpression of by several effectors from bacterial as well as fungal pathogens, See1, as they have an early ﬂoral switch (15 d) and are suitable for early utilizing it according to the need of the pathogen lifestyle. This meristematic tassel infections. Both cultivars of maize exhibit similar supports the early study of evolutionarily different effectors tar- infection symptoms. Both varieties were grown in T-type soil (FrühstorferPikiererde). Ustilago maydis strains were grown in YEPS (0.4% yeast geting a common host defense protein (Song et al., 2009) and is extract, 0.4% peptone, and 2% sucrose) at 28°C with shaking at 200 rpm also consistent with the model of evolutionarily different virulence of 0.6 to 0.8. Cells were centrifuged at 900g for 5 min, re- effectors targeting conserved hubs in a plant immune system suspended in water to OD of 1.0, and injected into stems of 7-d-old network (Mukhtar et al., 2011). Work to date demonstrated that maize seedlings with a syringe, as described previously (Kämper et al., SGT1 is involved in resistance during biotrophic interactions. The 2006). All infection assays were performed in three independent infection results reported here indicate that U. maydis alters SGT1 function trials with multiple seedlings. Disease symptoms were scored at 12 DPI Tumor Induction by Ustilago maydis using a previously developed scoring scheme (Kämper et al., 2006).
and quick penetration of the ﬁxer and allows better preservation of mitotic Tassel infections were done after 15 d of seed sowing in Gaspe Flint and stages (Kotogány et al., 2010).
after 4 weeks in Early Golden Bantam, as described previously (Walbotand Skibbe, 2010). Disease symptoms in the tassels were scored 14 DPI Yeast Transformation and Two-Hybrid Interaction Assay following the criteria described in a previous study (Schilling et al., 2014).
Nicotiana benthamiana plants (BN3) were grown in a phytochamber To identify host interactors of See1, See1 lacking the signal peptide was (Vötsch) under controlled environmental conditions (21°C, 16 h of light, 8 h expressed from pGBKT7-See1 and screened against a maize cDNA of dark) as described previously (Talarczyk et al., 2002).
library in pGADT7. Preliminary testing indicated that See1 was nontoxic toyeast (Saccharomyces cerevisiae) and did not autoactivate. The yeasttwo-hybrid library analyses were done using a normalized cDNA library of RNA Extraction and RT-qPCR infected maize tissue containing seedling and tassel samples. The strain Expression of the candidate gene was analyzed by RT-qPCR. Total RNA AH109 (Clontech) was used for all yeast assays unless otherwise men- was isolated from infected samples (leaf, tassel base, or anthers). The tioned. Yeast transformation was done as described in the DUAL infected plant material was collected at successive time points from 2 to membrane starter kit manual (Dualsystems Biotech). The yeast two-hybrid 14 DPI. To conﬁrm the high expression of see1 at 9 DPI, seedlings, anthers, screen was performed following the instructions of the Matchmaker and tassel base were collected from plants infected with U. maydis strain yeast two-hybrid manual (Clontech) using 1 mg of bait DNA (pGBKT7- Ppit2-see1 and the SG200 control. The samples were taken in three in- See1) and 0.5 mg of library DNA. All resulting yeast clones were tested by dependently conducted experiments. RNA was isolated using Trizol re- immunodetection for expression of the respective proteins. To perform agent (Invitrogen) and puriﬁed using an RNeasy kit (Qiagen). For cDNA a yeast dilution assay, 3 mL of selective medium (SD-Leu-Trp) was in- synthesis, the SuperScript III ﬁrst-strand synthesis SuperMix kit (Invitrogen) oculated with a single colony of the respective yeast strain and incubated was used to reverse transcribe 1 mg of total RNA with oligo(dT) primer. The overnight at 28°C. OD was adjusted to 0.2, and the cells were grown to RT-qPCR analysis was performed using an iCycler machine (Bio-Rad) in of 0.6 to 0.8. Next, 1 mL of yeast culture was centrifuged for 10 combination with iQ SYBR Green Supermix (Bio-Rad). Cycling conditions min at 3500g, and the pellet was washed twice with 1 mL of sterile water were 2 min at 95°C followed by 45 cycles of 30 s at 95°C, 30 s at 61°C, and ﬁnally resuspended in 500 mL of sterile water. OD and 30 s at 72°C. Gene expression levels were calculated relative to 1.0 with sterile water, and 5 mL of this suspension, as well as 1:10, 1:100, the peptidylprolyl isomerase gene (ppi ) of U. maydis for quantifying and 1:1000 dilutions, were applied on SD-Leu-Trp plates (low stringency) see1 expression (van der Linde et al., 2012). Error bars in all ﬁgures that as a growth control and SD-Leu-Trp-Ade-His plates (high stringency) to show RT-qPCR data represent the SD that was calculated from the test for protein-protein interaction. Growth was scored after 4 to 5 d of original cycle threshold values of three independent biological repli- incubation at 28°C.
cates. Primer sequences used for RT-qPCR are listed in For the yeast complementation assay of the yeast sgt1 mutants, yeast strains YKK 57 (sgt1-5) and YKK65 (sgt1-3) were transformed with con-structs to express maize SGT1 and the phosphovariants of SGT1 (cloned EdU-Based DNA Synthesis and Cell Proliferation Assay into pGREG536 vector under the control of the Gal1 promoter). Thetransformation was done as described previously, and the transformants Published protocols were used for U. maydis seedling infections (Kämper were selected on SC-Ura-2% (w/v) glucose plates. The strains were then et al., 2006) and tassel infections as described (Walbot and Skibbe, 2010).
shifted to SC-Ura-2% (w/v) galactose and incubated for 4 d to test their At 4 DPI, the third infected leaf where the ﬁrst infection symptoms ap- ability to complement the temperature-sensitive sgt1-5 and sgt1-3 growth peared was used for the EdU assay and incubated for 5 h with 10 mM EdU defects. Also, the transformants selected were serially diluted 5-fold (Invitrogen) in small chambers designed for labeling physiologically active for a drop assay and incubated at 25 and 37°C for 4 d to check for leaves. For the tassels at 3 DPI, the immature mitotically active tassel was bathed with 1 mL of 20 mM EdU after delivery with a 26-gauge hypodermicneedle through the whorl of leaves surrounding the inﬂorescence apex ;17 d after germination in the Gaspe Flint maize variety. EdU seeps intospikelets through the small air spaces between the external organs of the Confocal images were taken on a TCS-SP5 confocal microscope (Leica) spikelet and ﬂorets and reaches anthers over the labeling time of 5 h. After as described previously (Doehlemann et al., 2009). Details of the AF488 the labeling procedure, the area in seedlings below the infection holes was WGA and PI microscopy are given in . Fluores- detached and ﬁxed in 100% (v/v) ethanol. For the tassel tissue, around cence of AF488 alone or coupled to EdU was elicited at 488 nm and 150 anthers from different parts of the tassel were dissected to ensure detected at 495 to 540 nm. PI was excited at 561 nm and detected at 570 random sampling with equal probability of labeled anthers and ﬁxed in to 640 nm. YFP was excited at 514 nm and detected at 520 to 540 nm, 100% (v/v) ethanol 5 h after labeling, as done for seedlings. The EdU mCherry ﬂuorescence was excited at 561 nm and detected at 590 to 630 staining procedure was done as described previously (Kelliher and nm, and cell wall autoﬂuorescence used excitation of 405 nm and de- Walbot, 2011). The samples were washed once with fresh 100% (v/v) tection at 435 to 480 nm.
ethanol followed by two washes in PBS, pH 7.4, plus 2% (w/v) BSA, then Preparation of samples for TEM and immunogold labeling was per- the samples were transferred to permeabilization solution (PBS +1% [v/v] formed as described previously (Heyneke et al., 2013). Small samples Triton X-100) at room temperature for 20 min with rocking. After per- (;1.5 mm2) from at least 12 different leaves were cut on a modeling wax meabilization, samples were washed twice in PBS plus 2% (w/v) BSA and plate in a drop of 2.5% (w/v) paraformaldehyde and 0.5% (v/v) glutar- then directly incubated for 30 min at room temperature with EdU Click-IT aldehyde in 0.06 M Sørensen phosphate buffer, pH 7.2. Samples were cocktail for detection (Invitrogen) and 20 mg/mL PI (Molecular Probes) then ﬁxed for 90 min in the same ﬁxing solution. Samples were rinsed in directly added to the staining solution. The addition of EdU detection buffer four times for 15 min and dehydrated in increasing concentrations solution was done according to the manufacturer's instructions. The of acetone (50, 70, and 90% [v/v]) for 20 min at each step. Subsequently, samples were then washed twice in PBS, pH 7.4, plus 2% (w/v) BSA, specimens were gradually inﬁltrated with increasing concentrations of LR- transferred to PBS, pH 7.4, and kept at 4°C in the dark for several days White resin (30, 60, and 100% [w/v]; London Resin) mixed with 90% (v/v) before imaging. Triton X-100 treatment results in a nuclear stain and is acetone for a minimum of 3 h per step. Samples were ﬁnally embedded in compatible with EdU costaining. Moreover, it prevents the cell shrinkage pure, fresh LR-White resin and polymerized at 50°C for 48 h in small plastic containers under anaerobic conditions. Ultrathin sections (80 nm) Results) were inﬁltrated into 4-week-old N. benthamiana leaves as de- were cut with a Reichert Ultracut S ultramicrotome (Leica Microsystems).
scribed earlier. Expression of the Nb-MEK2 variants was induced with Immunogold labeling of See1-3xHA was done with ultrathin sections 30 mM DEX 40 to 48 h later (Yang et al., 2001). Treated leaves were collected on coated nickel grids with the automated immunogold labeling system ;5 h after DEX inﬁltration. Ground leaf material was thawed in 10 mL of Ex- Leica EM IGL (Leica Microsystems). The ideal dilutions and incubation strep buffer (100 mM Tris-HCl, pH 8.0, 5 mM EGTA, 5 mM EDTA, 150 mM times of the primary monoclonal anti-HA antibody (produced in mouse by NaCl, 10 mM DTT, 0.5 mM 4-(2-aminoethyl) benzenesulfonyl ﬂuoride hy- Sigma-Aldrich) and secondary antibodies (goat anti-mouse antibodies drochloride, 5 mg/mL antipain, 5 mg/mL leupeptin, 50 mM NaF, 1% [v/v] from British BioCell International) were determined in preliminary studies Phosphatase Inhibitor Cocktail 1 [Sigma-Aldrich], 0.5% [v/v] Triton X-100, by evaluating the labeling density after a series of labeling experiments.
and 100 mg/mL avidin) as described previously (Witte et al., 2004). The slurry The ﬁnal dilutions of primary and secondary antibodies used in this study was centrifuged for 10 min at 4°C (15,000g), the supernatant was ﬁltered showed a minimum background labeling outside the sample with a through Miracloth, and 0.5 mL of StrepTactin Sepharose (IBA) was added.
maximum speciﬁc labeling in the sample. The sections were blocked for Binding was performed by incubation of this suspension on a rotator for 1 h 20 min with 2% (w/v) BSA (Sigma-Aldrich) in PBS, pH 7.2, and then at 4°C. The slurry was transferred into a Poly-Prep column (Bio-Rad), and treated with the primary antibody against See1 3xHA diluted 1:2000 in the ﬂow-through was discarded. The resin was washed twice with 10 mL of PBS containing 1% (w/v) BSA. After section washing with PBS containing W-buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mM EDTA). Four 1% (w/v) BSA three times for 5 min, each grid was treated with 10-nm times 250 mL of E-buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM gold-conjugated secondary antibodies (goat anti-mouse IgG) diluted EDTA, and 2.5 mM desthiobiotin) was added, and eluates were collected.
1:100 in PBS containing 1% (w/v) BSA for 90 min at room temperature.
The samples were concentrated on a Microcon YM-10 (Millipore) for 30 min After a short wash in PBS (three times for 5 min) and distilled water (two at 4°C (13,000g) to a volume of 20 mL and resolved by SDS-PAGE.
times for 5 min), labeled grids were poststained with 2% uranyl-acetateaqueous solution for 15 s and then investigated with a Philips CM10 TEM Recombinant Protein Preparation apparatus. Micrographs of randomly photographed immunogold-labeledsections were digitized, and gold particles were counted automatically The full-length cDNA of maize SGT1 was cloned into the pET-15b vector.
using the software package Cell D with the particle analysis tool The full-length cDNA of SIPK was cloned into the pGEX-6P-1 vector. The (Olympus, Life and Material Science Europa) in different visually identiﬁed full-length cDNA of tobacco (Nicotiana tabacum) MEK2DD was cloned into and manually traced cell structures. The obtained data were statistically the pGEX-6P-2 vector. Escherichia coli (BL21) cells were induced with 0.5 evaluated using Statistica (Stat-Soft Europe).
mM isopropyl b-D-1-thiogalactopyranoside for Zm-SGT1 at 28°C for 4 hand with 0.25 mM isopropyl b-D-1-thiogalactopyranoside for SIPK and Coimmunoprecipitation of See1 and SGT1 in N. benthamiana MEK2DD at 18°C for 4 h. His-tagged recombinant protein was puriﬁed usingNi-NTA resin (Qiagen). Glutathione S-transferase-tagged recombinant proteins were puriﬁed using the Glutathione Agarose resin (Sigma-Aldrich).
YFP-HA were heterologously expressed in N. benthamiana. As ex-pression controls, the constructs were separately expressed with the Targeted Site-Directed Mutagenesis of Maize SGT1 appropriate empty vector. For all experiments, Agrobacterium tumefa-ciens GV3101 was transformed as described previously (Flowers and The targeted exchange of one or more bases in the plasmids was per- Vaillancourt, 2005). The transformants were inﬁltrated into N. ben- formed by PCR using the QuikChange Multi Site-Directed Mutagenesis kit thamiana leaves (3 to 4 weeks old) according to Sparkes et al. (2006). Four (Stratagene). The protocol used for this mutation and base substitution days after inﬁltration, the leaves were harvested and ground in liquid was according to the manufacturer's instructions. Up to three oligonu- nitrogen. The ground powder was mixed with buffer (50 mM Tris-HCl and cleotides were designed for the ampliﬁcation of the entire plasmid, with 150 mM NaCl, pH 7.0). The resulting leaf extract was centrifuged at 3500 one oligonucleotide containing the corresponding mutation(s) in the primer rpm at 4°C and subsequently sterile-ﬁltered. The protein concentration of the extract was determined with the Roti-Quant Protein quantiﬁcationassay (Carl Roth). To 1 mL of leaf extract containing 2 mg/mL protein, 50m In Vitro Phosphorylation Assays L of anti-HA Afﬁnity Matrix (Roche Diagnostics) was added, and samples were incubated overnight at 4°C on a rotation wheel. The samples were The puriﬁed recombinant 6xHis-Zm-SGT1 or 6xHis-Zm-SGT1AP (5 mg then centrifuged through Pierce SpinColumns (Thermo Scientiﬁc) and each) was incubated at 30°C for 30 min with SIPK and MEK2DD (2 mg each) washed once with buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.0), and in the reaction buffer (20 mM Tris-HCl, pH 7.5, 40 mM MgCl , and 10 mM protein was ﬁnally eluted by boiling samples in 23 SDS loading buffer for 5 EGTA) or with crude plant protein extracts from the infected maize samples min. Appropriate amounts of the eluted proteins were separated by SDS- of seedling leaves and tassel base with U. maydis wild-type SG200, PAGE, followed by transfer to a nitrocellulose membrane. After electro- SG200Dsee1, or the overexpression strain with See1 (approximately 20 mg blotting, the membrane was saturated with 5% (w/v) nonfat dry milk in each) in the reaction buffer (40 mM Tris-HCl, pH 8.0, 10 mM MgCl , 1 mM TBS-T (50 mM Tris-HCl, 150 mM NaCl, pH 7.6, and 0.1% [v/v] Tween 20) DTT, and 0.1% [v/v] Triton X-100). Both buffers contained 50 mM ATP and for 1 h at room temperature. After blocking, the membrane was washed 1.5 mCi of [g-32P]ATP. The reaction was terminated by adding 33 Laemmli three times with TBS-T followed by incubation with the primary antibody sample buffer.
(anti-HA antibody, 1:10,000; anti-c-Myc antibody, 1:5000; Sigma-Aldrich)overnight at 4°C. Membranes were washed three times prior to incubation Mass Spectrometry Analysis for 1 h with horseradish peroxidase-conjugated secondary antibody (anti-mouse antibody, 1:5000; Cell Signaling). Signals were detected using Gel bands containing the proteins of interest were subjected to a standard SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientiﬁc).
proteomic procedure as described in Brieﬂy,reduced and alkylated proteins were subjected to trypsin digestion. Theresulting peptides were eluted from the gel, and phosphopeptide enrich- In Planta Phosphorylation Assay ment was performed on SwellGel Gallium-Chelated Discs (Phosphopeptide The constructs encoding maize SGT1 in the presence or absence of See1 Isolation Kit; Thermo Scientiﬁc). Liquid chromatography-mass spectrometry and also in addition to the earlier mentioned controls (described in analyses of the peptide mixtures were performed on the Orbitrap Velos Tumor Induction by Ustilago maydis spectrometer (Thermo Scientiﬁc). The Mascot program was used for AF, AF494083.1; tobacco SIPK, AF165186; tobacco MEK2, AF325168; database searches, and tandem mass spectrometry spectra of phos- maize MAPK kinase 2, NP_001104843.1; maize putative MAPK family phorylated peptides were also curated manually.
protein, AFW85791.1; maize putative MAPK MPK6, ACG37232.1; maize ABAstimulation MAPK, NP_001152745.1; maize unknown kinase, ACF85409.1.
Microarray Analysis Supplemental Data For the microarray experiments, maize plants (cv Early Golden Bantam) Scoring Scheme for Leaf Tumors Based on were grown in a phytochamber with the same climatic conditions as Symptoms Observed at 12 DPI.
described previously. Plants were inoculated in three independent bi-ological replicates with water (mock), SG200, and SG200Dsee1 as de- SG200Δsee1 Mutant Completes Its Entire scribed previously in virulence assays for seedling infections (Kämper et al., 2006; Doehlemann et al., 2008). Infected or mock-inoculated tissue Formation of Ear Tumors in SG200Δsee1 Mutant from 15 plants per treatment was harvested at 6 DPI by excising a section Demonstrates the Independence of Floral Tumors on See1 Activity.
of the third leaf between 1 and 3 cm below the injection holes. For RNAextraction, material from 15 plants per treatment was pooled and ground Growth of SG200Δsee1 Mutant Is Arrested in to powder in liquid nitrogen, and RNA was extracted with Trizol reagent the Mesophyll and Vascular Cell Layers of Leaves.
(Invitrogen). RNA was puriﬁed using the RNeasy kit (Qiagen). Agilent Gene Expression during Maize Colonization 4x44k maize genome arrays were used in three biological replicates, using with SG200 and SG200Δsee1.
the standard Agilent One Color Microarray-based gene expression Speciﬁcity of the EdU Labeling Assay.
analysis low-input quick Amp labeling protocol. Expression data weresubmitted to the Gene Expression Omnibus See1 Requirement in Leaf Tumor Formation.
accession number GSE63077). For further experimental details, Constitutive Overexpression of See1 Results in Tumors on the Vegetative Parts of Tassels.
Stable Expression of the Ppit2 overex- Plasmid Constructs and Nucleic Acid Construction pressed See1-HA Protein in Infected Seedlings and Tassel Base.
For plasmid construction, standard molecular cloning strategies and Overexpression of See1 Results in Two techniques were applied (Sambrook et al., 1989). All plasmids generated Abnormalities in Tassels: Tumors at the Tassel Base and Greenish and used in this study are listed in Oligonucleotides that were used for PCR are shown in All restrictionenzymes used in this study were purchased from New England Biolabs. For See1 Is Transferred to Cells Neighboring to a detailed description of plasmid constructs, see .
the Transformed Cell.
The isolation of genomic U. maydis DNA was performed as described Stable Expression of the See1-3xHA Protein previously (Schulz et al., 1990). All U. maydis strains ( in Infected Tissues.
are derived from SG200 and were generated by insertion of p123 See1 Is Translocated to the Plant Cell derivatives into the ip locus as described (Loubradou et al., 2001). Isolated Cytoplasm and Nucleus.
U. maydis transformants were tested by DNA gel blot hybridization toassess single or multiple integration events in the ip locus. For all the Complementation of Two Yeast sgt1 Cell cloning work, PCR was performed using Phusion High-Fidelity (New Cycle Temperature Sensitive Mutants with Maize SGT1.
England Biolabs). The PCR products of the different genes were cleaned The Deduced SGT1 Protein Sequences of up before digestion using the Wizard SV Gel and PCR Clean-Up System Z. mays and N. benthamiana Showing a Multiple Alignment.
(Promega). The vectors were transformed into either DH5a or Top10 cells(Invitrogen) and then plated on YT-agar plates containing a speciﬁc se- The Deduced SIPK Protein Sequences of lection marker. Plasmids were extracted using the QIAprep system N. tabacum and a Set of Five Putative Z. mays SIPK Showing (Qiagen). All plasmid constructs containing ampliﬁed gene fragments a Multiple Alignment.
were sequenced.
Expression Level of the Nt-MEK2, Nb-Zm-SGT1, and Um-See1 Transiently Expressed in N. benthamiana Leaves Bioinformatics Tools Applied in This Study to Assess Zm-SGT1 Phosphorylation in Planta.
The Deduced SGT1 Protein Sequences with Signal peptide prediction was performed with the online program SignalP Marked Phosphorylation Sites within Zm-SGT1.
4.1 (Protein conserved domainsearch was performed with the online program Pfam Salicylic Acid Induced Protein Kinase The mass spectrometry data were analyzed using Mascot Phosphorylates Zm-SGT1 in Vitro.
Distiller software (version 2.1.1; Matrix Science) and compared with the Subcellular Localization of the Phosphovar- NCBInr database using the Mascot database search engine (version 2.1; iants of Maize SGT1.
Matrix Science), as described in detail in Complementation of Two Yeast sgt1 CellCycle Temperature-Sensitive Mutants with Maize SGT1 Wild Type, Accession Numbers Phosphomimic, and Phosphonull.
Sequence data from this article can be found in the GenBank/EMBL data In Planta Phosphorylation of Maize SGT1AP.
libraries under the following accession numbers: U. maydis see1, Differentially Expressed Top GO Terms XP_758386.1; U. maydis pit2, XP_757522.1; U. maydis ppi, XM_754780.1; Related to the DNA Synthesis and Cell Differentiation.
maize SGT1.1 of cv Early Golden Bantam, KP789376; maize SGT1.2,ACF84496.1; N. benthamiana SGT1 AY, AY899199.1; N. benthamiana SGT1 Oligonucleotides Used in This Study.
Plasmids Used in This Study.
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