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Received Date : 23-May-2014 Revised Date : 28-Jul-2014 Accepted Date : 18-Sep-2014 Article type

: Research Letter

Editor

: Skorn MONGKOLSUK

Small heat shock proteins (HSP12, HSP20 and HSP30) play a role in Ustilago maydis pathogenesis Anupama Ghosh Division of Plant Biology, Centenary campus, P1/12 C.I.T. Road Scheme VII M, Kolkata 700054, West Bengal, India Correspondence: [email protected]

Keywords: Ustilago maydis, heat shock protein, pathogenicity, Zea mays, oxidative stress

ABSTRACT Small heat shock proteins (HSP) play multiple functions within a cell. These functions primarily include regulation of growth and survival in response to different stresses. However in some cases small HSPs have been shown to play crucial roles in microbial pathogenesis. Ustilago maydis genome also codes for a number of small HSPs. In the present study we elucidate the role of U maydis small HSPs in the pathogenicity as well as general stress response of the fungus. Through quantitative real time PCR analysis the expression levels of small HSP genes in comparison to other HSPs were accessed both during infection of the host plant Zea mays as well as when the pathogen is subjected to an abiotic stress like oxidative stress. This study revealed that in contrary to other HSPs small HSPs showed an increased level of differential expression under both the tested conditions indicating towards This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12605 This article is protected by copyright. All rights reserved.

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a possible role of small HSPs in the pathogenicity and stress response of U. maydis. This has been further confirmed by generation of deletion and complementation strains of three putative small HSPs. INTRODUCTION Heat shock proteins comprise of a family of molecular chaperones that aid in the stabilization and correct folding of nascent polypeptides (Ellis & Hartl, 1999). Besides, HSPs also play important roles in the maintenance of total cellular proteome both under physiological conditions and under conditions of stress. For instance HSPs have been found to be associated with different cellular functions like assembly of macromolecular complexes, protein transport and dissociation and refolding of aggregates of stress-denatured proteins (Hartl, 1996, Kim, et al., 2013). In addition to these HSPs are also involved in some specialised functions like transcriptional control within a cell (Pirkkala, et al., 2001) and control of programmed cell death (Garrido, et al., 2001). Invasion of host tissue by a pathogen is another such situation where both the host and the pathogen get exposed to different damaging stress stimuli like for instance degradative enzymes, different reactive radicals and extreme pH. A very interesting question that can be raised at this point is whether HSPs are activated under these stressed conditions to impart protective measures towards both the host and the pathogen. Several studies during the past few decades have clearly shown the involvement of host HSPs in the defence response against invasion by a pathogen (Liu, et al., 2014). In the pathogen side however most of the studies were carried out in pathogenic bacteria where an increased expression of HSPs could be linked to the infection of their respective hosts (Buchmeier & Heffron, 1990, Gahan, et al., 2001, Monahan, et al., 2001, Gaywee, et al., 2002, Schnappinger, et al., 2003). Similar studies in pathogenic fungi are mostly lacking. However, a very interesting finding in this direction is the involvement of Magnaporthe oryzae HSP70 family proteins LHS1 and KAR2 in the pathogenicity of the fungus towards the host plant rice (Yi, et al., 2009). Besides, a recent study led by Becherelli M and colleagues have shown the requirement of HSPs for the development of biofilms in Candida albicans during infection (Becherelli, et al., 2013). In addition to these some reports are also available showing the involvement of major heat shock protein HSP 90 in the stress response and pathogenesis of fungi like S. cereviseae Candida albicans and Aspergillus fumigatus (Singh, et al., 2009, Robbins, et al., 2011, Lamoth, et al., 2012, Robbins, et al., 2012). Besides these major HSPs, some of the small HSPs have also been found to undergo differential expression in response to various stress This article is protected by copyright. All rights reserved.

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conditions in fungi. For instance S. cereviseae small HSP, HSP12 has been shown to be required for the growth and survival of the fungus under a variety of stress conditions and also for the maintenance of normal cell morphology (Welker, et al., 2010). Involvement of small HSPs in microbial pathogenicity however has been evidenced primarily in bacteria like Mycobacterium tuberculosis and Agrobacterium tumifaciens (Stewart, et al., 2005, Tsai, et al., 2009, Tsai, et al., 2010). In fungi a similar example is available in case of Candida albicans where a small HSP, HSP21 has been found to be involved in the adaptation of the fungus in specific environmental stresses, immune evasion and pathogenicity (Mayer, et al., 2012). Ustilago maydis is a basidiomycetous fungus that causes smut disease of maize. The lifecycle of the pathogen comprises of a non-infectious sporidial form and an infectious hyphal form. The development of the infectious form is dependent on the physical association of compatible mating pairs of the fungi with the plant surface that leads to the progression of the infection (Brefort, et al., 2009). Although a number of studies have already been done to elucidate the mechanism of pathogenic development of the fungus (Heimel, et al., 2013), nothing is known about the involvement of HSPs if any in the virulence of the fungus. However studies led by Holden D W (Holden, et al., 1989) and Salmeron-Santiago K G (Salmeron-Santiago, et al., 2011) showed differential expression of certain HSP 70 family proteins in response to different stress conditions involving heat stress and osmotic stress. We therefore in the present study tried to explore the status of U. maydis heat shock proteins, small HSPs in particular during an event of maize infection using mating compatible FB1 and FB2 strains of the fungus. Within the scope of the present study we also attempted to compare the expression patterns of small HSPs with that of other HSPs like HSP 70 that has an established involvement in stress response and pathogenesis in many other systems during pathogenesis. Besides we also tried to access the expression patterns of the respective HSPs when the fungus is subjected to an abiotic stress like oxidative stress induced by hydrogen peroxide. For this study however we used a solopathogenic strain (SG200) of the fungus. A comparison of the above two conditions indicated that U.maydis small heat shock proteins (Um00205, Um03881, Um04125) probably play important role in the general stress response by the fungus irrespective of its nature whether biotic or abiotic.

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MATERIALS AND METHODS Strains U. maydis strains FB1, FB2 (Banuett & Herskowitz, 1989), SG200 (Kamper, et al., 2006) and their derivatives are used throughout the study. Maize variety Early Golden Bantam (EGB) was used for infection studies. For the cloning purposes E. coli strain DH5α was used. All strains used in this study are listed in table S1. U. maydis strains were generated by insertion of p123 derived plasmids into the ip locus of U. maydis genome as described previously (Loubradou, et al., 2001). For generation of deletion strains the PCR strategy described by Kamper (Kamper, 2004) and the SfiI insertion cassette system

was used

(Brachmann, et al., 2004). The transformants were confirmed by PCR and sequencing of the altered genetic loci. Plasmid constructions Standard molecular cloning techniques were used to generate all the plasmids used in this study. The cloning strategies and respective primers used are listed in table S2 and S3 respectively. Plant infections For plant infections desired strains of U. maydis were grown in YEPSL media (0.4% yeast extract, 0.4% tryptone, 2% sucrose) until the OD600 reached 0.8. The cells were then harvested and resuspended in sterile water to a final OD600 of 1.0. The resulting suspensions of U. maydis cells were used for infecting 7 days old seedlings of EGB using syringe infection. For FB1, FB2 infections equal volumes of each of the two cell suspensions were mixed before seedling injections. Disease symptoms were scored 12 days post infection as per the disease rating criteria described by Kaemper et al (Kamper, et al., 2006). Three independent experiments were performed for each infection and the average values were expressed as percentage of the total number of infected plants. Hydrogen peroxide treatment of U. maydis strains For treatment of U. maydis strains with H2O2, indicated strains were grown in YEPSL media till OD600 reached 0.8 following which the cells were treated with desired concentrations of H2O2 for 30 min. Following this incubation step the cells were washed with PBS and resuspended in fresh YEPSL media. The cells were then incubated in this media for indicated This article is protected by copyright. All rights reserved.

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period of time. For quantitative real time analysis cells were collected just after the 30 min incubation with H2O2. Colony forming unit assay 1x106 cells from either treated or untreated U. maydis strains were collected in 1 ml of YEPSL media. The resulting suspension was diluted to 1:1000 and from it 100 µl was spread on PD agar plates. The plates were incubated for 48 hrs and the number of colonies that appeared on the plates were counted. Quantitative real time PCR For quantitative real time PCR RNA was extracted from either axenically grown treated or untreated U. maydis strains or infected plant materials at indicated time points using TRIZOL reagent (Invitrogen) following manufacturer’s protocol. The extracted RNAs were treated with DNase (Ambion) and subsequently used for cDNA synthesis. cDNA synthesis was carried out using Superscript III first-strand synthesis SuperMix assay (Invitrogen) using 1 µg of total RNA. Quantitative RT-PCR was performed on a Bio-Rad iCycler using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Sequences of the primers used are listed in table S3. RESULTS U. maydis heat shock proteins U. maydis genome codes for several HSP genes. In the present study we attempted to investigate the contribution of small HSPs in particular in the pathogenesis as well as general stress response of U. maydis towards abiotic stresses like for instance oxidative stress induced by exposure to hydrogen peroxide. Accordingly three different genes were chosen from U. maydis genome database (available at http://mips.helmholtz-muenchen.de) representing three different families of small HSPs namely HSP12, HSP20 and HSP30. Through BLAST analysis against U. maydis protein database using Saccharomyces cereviseae HSP12 (NCBI accession CAA39306.1) as the query sequence we could identify U. maydis homologue Um00205. A similar analysis was carried out with the amino acid sequence of S. cereviseae HSP30 family protein YRO 2 (NCBI accession No. AHY74535.1) to identify U. maydis homologue Um04125. In order to identify HSP20 homologue of U. maydis however a separate approach was adopted. U. maydis protein database was searched for the presence of

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proteins containing alpha crystalline/HSP20 domain (Interpro accession: IPR002068). This resulted in the identification of Um03881 as U. maydis homologue of HSP20. The respective proteins were also found to be annotated as candidate HSP12, HSP30 and HSP20 homoloues in U.maydis genome database. In addition to these we also chose a small set of another three HSPs with molecular weights ranging from 40 kDa to 100 kDa to get a comparative overview of the behaviour of these HSPs with respect to small HSPs under similar conditions. Among these were included homologues of HSP 70, 60 and 100. A search in the U.maydis genome database for proteins possessing domains for HSP70 family (Interpro accession: IPR013126) revealed the presence of about 7 candidates. Out of these we selected during the initial course of experiments two candidate HSP70 proteins (Um10526 and Um01209). Um10526 represented a homologue of SSB2 family of cytosolic HSP70 and Um01209 represented SSC1 family of mitochondrial HSP70 homologue. However due to a lack of any change in the expression pattern of Um01209 during pathogenesis or oxidative stress under our experimental conditions (data not shown) we excluded this candidate from all further experiments and proceeded with Um10526 as one of the representatives of HSP70 family proteins in U. maydis. In order to get a homologue of HSP60 the U. maydis database was searched for proteins possessing conserved chaperonin60 domain (Interpro accession: IPR001844). This returned 8 hits out of which only one (Um05831) was found to be annotated as probable heat shock protein 60. Um05831 was therefore considered as representative of U. maydis HSP60 family proteins. U. maydis homologue of S. cereviseae HSP104, Um06430 was identified through BLAST P analysis on U. maydis protein database. S. cereviseae HSP104 sequence (NCBI aceession No. CAA97475.1) was used as the query sequence. Besides all these HSPs, HSP90 has been shown in many different systems to play important roles in different stress responses. We therefore tried to identify a HSP90 homologue in U. maydis genome database for the present study. A BLAST P analysis using S. cereviseae HSP90 family protein, HSP82 sequence (NCBI accession No. DAA11197) yielded only one hit (Um05134) with a very low expect value of 0.0093057. Because of such low homology Um05134 was excluded from present analysis. Expression profile of U.maydis HSPs during host infection HSPs play crucial roles in stress response in different organisms. Induced expression of HSPs in response to different stresses therefore is often observed in a wide variety of different organisms. In order to gain insights into the function of selected U. maydis HSPs and to investigate their possible role in stress response during an event of host invasion we carried This article is protected by copyright. All rights reserved.

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out quantitative real time analysis of HSP RNAs from infected plant samples at different time intervals. Our data showed that two of the selected small HSPs (Um00205 and Um04125) undergo an induction of expression in the early phase of infection, 1 day post infection (1dpi). In case of Um03881 however an induction of expression has been noticed at a relatively later time point i.e., 6dpi. In the category of regular HSPs except for Um06430 that shows an induction of about 140 fold compared to axenic culture, the other two HSPs i.e., Um05831 and Um10526 showed either no or very negligible induction of expression 1dpi (Fig. 1). All of the small HSPs also showed an induced expression at 12dpi although to different extents. Among the regular HSPs however the expression of Um06430 was found to be induced to about 50 folds at 12dpi compared to that of the axenic culture. Whereas Um05831 showed a very negligible (about 1.5 fold) increase in expression, Um10526 on the contrary showed a reduction in expression to about half of that in case of axenic culture. Taken together the data indicate that different HSPs probably play distinctive roles in the development of infection by the pathogen that is quite obvious from the temporal difference in their induction pattern. Small HSPs Um00205 and Um04125 most likely play key roles in the initial establishment of the infection. A similar function could also be presumed for Um06430. However from the obtained expression data it is very difficult to predict any involvement of Um05831 and Um10526 in the establishment and development of the disease by the pathogen. Moreover induction of the small HSP genes and Um06430 a second time at a later time point like 12 dpi may also indicate towards the involvement of these HSPs in the more advanced developmental stages of pathogen inside the host like proliferation and sporogenesis. U. maydis small HSPs Um00205, Um03881 and Um04125 contribute towards the virulence of the pathogen Based on the expression data of U. maydis HSP genes in axenic culture and infected plant tissue we hypothesised that U. maydis small HSPs might be involved in the pathogenicity of the fungus towards maize. In order to validate our hypothesis we generated U. maydis SG200 strains lacking either Um00205, Um03881 or Um04125 genes. An infection of maize plants with each of these strains independently revealed a significant loss of virulence in each case. Unlike wild type (WT) SG200 strain neither of the knockout strains could produce large tumors with stunted growth of the infected plants. Although the population of infected plants showing large tumors remained almost equal in case of both the WT as well as the knockout strains, the population showing small tumors and chlorosis was found to be much more in This article is protected by copyright. All rights reserved.

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case of knockout strains when compared to that of WT strain (Fig. 2). All together the data shows that the deletion of the above mentioned genes from U. maydis genome results in lowering the severity of infection caused by the pathogen. Moreover all the noticed differences in the pathogenicity of the deletion strains were found to be perfectly complemented upon expression of the respective genes within the knockout background of the respective strains (Fig. 2). U. maydis genes Um00205, Um03881 and Um04125 confer protection against oxidative stress to the pathogen induced by hydrogen peroxide After we saw an effect of U. maydis small HSPs in the pathogenicity of the fungus we were curious to know whether these genes also play a role in the defence response of the pathogen against abiotic stresses. We therefore subjected different strains of U. maydis to oxidative stress using different concentrations of H2O2. We found that as we increase the concentration of H2O2 from 0 to 5 mM the viability of different U. maydis strains were gradually reduced when accessed over a time period of 3 hours (Fig. 3). When we did the same assay with strains lacking any of the three small HSP genes we obtained a similar result that is decrease in the viability of the cells as the concentration of H2O2 is increased (Fig. 3). But interestingly in each case we found that the viability of the knockout strains were always less that of the WT strains irrespective of the concentration of H2O2 applied. This indicated that these small HSPs of U. maydis play a role in the defence response of the pathogen against oxidative stress and their absence makes the cells more prone to damage by H2O2. Expression profile of U. maydis HSPs during oxidative stress induced by H2O2 To further confirm our data on the effect of U.maydis small HSPs on the defence response of the pathogen against oxidative stress we carried out quantitative real time analysis of HSP RNA levels in H2O2 treated WT as well as knockout strains. Our data revealed an increased expression of all the HSP genes tested in response to H2O2 treatment (Fig. 4). However the level of induction of the small HSPs was found to be much higher compared to that of regular HSPs. For instance when treated with 2 mM H2O2 in case of Um00205, Um03881 and Um04125 the expression levels were found to be approximately 1200-1500 folds of that of untreated cells. However if we look at the expression levels of regular HSPs the increase is only in the range of 15-50 folds when treated with 2 mM H2O2. From this data it seems that small HSPs have greater contribution towards stress response of the pathogen against oxidative stress. This article is protected by copyright. All rights reserved.

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DISCUSSION Heat shock proteins play important roles in the stress response mechanisms of different organisms. The present study poses an example of the same. In this study we could show induction of expression of some of the tested HSPs especially from the small HSP category in response to host infection as well as oxidative stress. Irrespective of the time of their induction following plant infection all the small HSPs show an effect on the pathogenicity of U. maydis when their respective genes were deleted from the genome of the fungus. This probably indicates that different HSPs participate in different developmental stages of U. mayis that together contribute towards the overall pathogenicity of the same. U. maydis sporidial cells produce dikaryotic filaments after mating on the plant surface. The resulting hyphal form of the fungus undergoes several developmental stages inside the plant tissue untill it give rise to teliospores (Banuett & Herskowitz, 1996). The involvement of small HSPs in any of these in planta developmental stages of U. maydis remains elusive. Nevertheless several such instances of HSPs being involved in different developmental stages of fungal pathogens are available in the literature. For instance in Candida albicans an overexpression of a small heat shock protein HSP12 has been found to be associated with enhanced cell to cell adhesion and subsequent formation of germ tube (Fu, et al., 2012). Moreover the reduced ability of the U. maydis strains that are deficient in different small HSP genes to survive under conditions of oxidative stress also indicate the possible involvement of the respective proteins in the defense response of the pathogen against different abiotic stresses. Our data in the present study gives an indication towards a greater involvement of small HSPs compared to regular ones towards both the stress response against biotic or abiotic stresses. Further studies however are needed to elucidate the exact mechanism of action of different small HSPs with respect to both the pathogenicity of the fungus and its tolerance against an abiotic stress like oxidative stress. REFERENCES Banuett F & Herskowitz I (1989) Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proc Natl Acad Sci U S A 86: 5878-5882. Banuett F & Herskowitz I (1996) Discrete developmental stages during teliospore formation in the corn smut fungus, Ustilago maydis. Development 122: 2965-2976. Becherelli M, Tao J & Ryder NS (2013) Involvement of heat shock proteins in Candida albicans biofilm formation. J Mol Microbiol Biotechnol 23: 396-400. Brachmann A, Konig J, Julius C & Feldbrugge M (2004) A reverse genetic approach for generating gene replacement mutants in Ustilago maydis. Mol Genet Genomics 272: 216-226.

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Brefort T, Doehlemann G, Mendoza-Mendoza A, Reissmann S, Djamei A & Kahmann R (2009) Ustilago maydis as a Pathogen. Annu Rev Phytopathol 47: 423-445. Buchmeier NA & Heffron F (1990) Induction of Salmonella stress proteins upon infection of macrophages. Science 248: 730-732. Ellis RJ & Hartl FU (1999) Principles of protein folding in the cellular environment. Curr Opin Struct Biol 9: 102-110. Fu MS, De Sordi L & Muhlschlegel FA (2012) Functional characterization of the small heat shock protein Hsp12p from Candida albicans. PLoS One 7: e42894. Gahan CG, O'Mahony J & Hill C (2001) Characterization of the groESL operon in Listeria monocytogenes: utilization of two reporter systems (gfp and hly) for evaluating in vivo expression. Infect Immun 69: 3924-3932. Garrido C, Gurbuxani S, Ravagnan L & Kroemer G (2001) Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 286: 433-442. Gaywee J, Radulovic S, Higgins JA & Azad AF (2002) Transcriptional analysis of Rickettsia prowazekii invasion gene homolog (invA) during host cell infection. Infect Immun 70: 6346-6354. Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381: 571-579. Heimel K, Freitag J, Hampel M, Ast J, Bolker M & Kamper J (2013) Crosstalk between the Unfolded Protein Response and Pathways That Regulate Pathogenic Development in Ustilago maydis. Plant Cell 25: 4262-4277. Holden DW, Kronstad JW & Leong SA (1989) Mutation in a heat-regulated hsp70 gene of Ustilago maydis. EMBO J 8: 1927-1934. Kamper J (2004) A PCR-based system for highly efficient generation of gene replacement mutants in Ustilago maydis. Mol Genet Genomics 271: 103-110. Kamper J, Kahmann R, Bolker M, et al. (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444: 97-101. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M & Hartl FU (2013) Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82: 323-355. Lamoth F, Juvvadi PR, Fortwendel JR & Steinbach WJ (2012) Heat shock protein 90 is required for conidiation and cell wall integrity in Aspergillus fumigatus. Eukaryot Cell 11: 1324-1332. Liu HY, Dicksved J, Lundh T & Lindberg JE (2014) Expression of Heat Shock Protein 27 and 72 Correlates with Specific Commensal Microbes in Different Regions of Porcine Gastrointestinal Tract. Am J Physiol Gastrointest Liver Physiol. Loubradou G, Brachmann A, Feldbrugge M & Kahmann R (2001) A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis. Mol Microbiol 40: 719-730. Mayer FL, Wilson D, Jacobsen ID, et al. (2012) Small but Crucial: The Novel Small Heat Shock Protein Hsp21 Mediates Stress Adaptation and Virulence in Candida albicans. PLoS One 7. Monahan IM, Betts J, Banerjee DK & Butcher PD (2001) Differential expression of mycobacterial proteins following phagocytosis by macrophages. Microbiology 147: 459-471. Pirkkala L, Nykanen P & Sistonen L (2001) Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J 15: 1118-1131. Robbins N, Leach MD & Cowen LE (2012) Lysine deacetylases Hda1 and Rpd3 regulate Hsp90 function thereby governing fungal drug resistance. Cell Rep 2: 878-888. Robbins N, Uppuluri P, Nett J, et al. (2011) Hsp90 governs dispersion and drug resistance of fungal biofilms. PLoS Pathog 7: e1002257. Salmeron-Santiago KG, Pardo JP, Flores-Herrera O, Mendoza-Hernandez G, Miranda-Arango M & Guerra-Sanchez G (2011) Response to osmotic stress and temperature of the fungus Ustilago maydis. Arch Microbiol 193: 701-709. Schnappinger D, Ehrt S, Voskuil MI, et al. (2003) Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment. J Exp Med 198: 693704.

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Singh SD, Robbins N, Zaas AK, Schell WA, Perfect JR & Cowen LE (2009) Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin. PLoS Pathog 5: e1000532. Stewart GR, Newton SM, Wilkinson KA, et al. (2005) The stress-responsive chaperone alpha-crystallin 2 is required for pathogenesis of Mycobacterium tuberculosis. Molecular Microbiology 55: 11271137. Tsai YL, Chiang YR, Narberhaus F, Baron C & Lai EM (2010) The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer. Journal of Biological Chemistry 285: 19757-19766. Tsai YL, Wang MH, Gao C, Kluesener S, Baron C, Narberhaus F & Lai EM (2009) Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in Agrobacterium tumefaciens. Microbiology-Sgm 155: 3270-3280. Welker S, Rudolph B, Frenzel E, et al. (2010) Hsp12 Is an Intrinsically Unstructured Stress Protein that Folds upon Membrane Association and Modulates Membrane Function. Molecular Cell 39: 507-520. Yi M, Chi MH, Khang CH, Park SY, Kang S, Valent B & Lee YH (2009) The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae. Plant Cell 21: 681-695.

FIGURE LEGENDS Fig. 1. Quantitative real time expression analysis of U. maydis HSP genes. Expression of U. maydis HSP genes Um00205, Um03881, Um04125, Um05831, Um06430 and Um10526 during different life cycle stages of the pathogen (axenic culture, 1dpi, 2dpi, 4dpi, 6dpi and 12dpi) was quantified using real-time PCR. Constitutively expressed peptidyl prolyl cis trans isomerase (ppi) was used for normalisation. Transcript level of the indicated genes axenic culture was considered to be 1. In all the other stages the expression level of individual HSP genes is shown as a fold change compared to that of the respective genes in axenic culture. Error bars represent standard deviation calculated from three independent biological replicates. Fig. 2. Pathogenicity assay with U. maydis deletion strains SG200∆Um00205, SG200∆Um03881, SG200∆Um04125 and complementation strains SG200∆Um00205Um00205-HA, SG200∆Um03881-Um03881-HA, SG200∆Um04125-Um04125-HA. Zea mays plants were infected with the indicated strains of U. maydis. Disease symptoms were scored 12 dpi and compared with that developed from infection with wild type SG200 strain. Disease symptom categories are depicted on right. Mean value of the data from three independent infections is shown in case of each strain. Error bars represent standard deviation. Fig. 3. Effect of H2O2 on the viability of different U. maydis strains. U. maydis SG200 strains either wild type or deficient in different HSP genes like Um00205, Um03881 and Um04125

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were exposed to 0-5 mM H2O2 for three hours. The percent survival rate of each of the strains either treated or untreated at indicated concentration of H2O2 was calculated. Mean value of three independent experiments was plotted against respective concentration of H2O2. Error bars represent standard deviation. Fig. 4. Oxidative stress induces expression of U. maydis HSP genes. Real time PCR was used to quantify expression levels of different U. maydis HSP genes Um00205, Um03881, Um04125, Um05831, Um06430 and Um10526 in U. maydis SG200 strain subjected to 0-5 mM H2O2 for 30 min. Constitutively expressed peptidyl prolyl cis trans isomerase was used for normalisation. Transcript level of the indicated genes in 0 mM H2O2 treated SG200 strain was considered to be 1. In all the other treated samples the expression level of individual HSP genes is shown as a fold change compared to that of the respective genes in 0 mM treated SG200 strain. Error bars represent standard deviation calculated from three independent biological replicates.

ACKNOWLEDGEMENTS I would like to thank Prof. Regine Kahmann for providing me with the U. maydis strains SG200, FB1 and FB2. I am also thankful to Prof. Sampa Das for providing some of the reagents used in this study and also for providing lab space to carry out a part of the project. This work is partially supported by the INSPIRE Faculty Scheme grant, IFA-13 LSPA-16.

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Small heat shock proteins (HSP12, HSP20 and HSP30) play a role in Ustilago maydis pathogenesis.

Small heat shock proteins (HSP) have multiple functions within a cell. These functions primarily include regulation of growth and survival in response...
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