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Keywords:

  • biotrophic interaction;
  • brome mosaic virus;
  • Ustilago maydis;
  • virus-induced gene silencing;
  • Zea mays

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression.
  • To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout.
  • In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis.
  • The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Maize (Zea mays spp. mays) is one of the world’s most important crop plants, being the major source for the production of lifestock feed and bio-ethanol. In addition to its agronomic importance, maize has been a leading model organism for basic plant research for almost a century (Strable & Scanlon, 2009). Significant losses in annual corn production are caused by fungal pathogens, and > 70 fungal diseases have been described for maize (Shurtleff et al., 1993). With > 1500 described species, the basidiomycete smut fungi are one of the largest groups of plant pathogenic fungi, causing significant losses in crop production world-wide (Martinez-Espinoza et al., 2002). Because of its excellent accessibility to reverse genetic methods, the causative agent of maize smut, Ustilago maydis, has become a model system for biotrophic fungi (Kämper et al., 2006). Basically all aerial parts of maize plants can be infected and after infection are transformed into tumor-like structures, in which U. maydis completes its life cycle (Brefort et al., 2009; Skibbe et al., 2010). In a previous study we examined transcriptional and metabolic changes in maize leaves during a compatible U. maydis interaction. Microarray hybridizations showed the induction of an unexpectedly high number of defense-related genes at the early stage of interaction 12 h after infection. At this time-point, fungal hyphae developed on the leaf surface and began to penetrate the epidermis (Doehlemann et al., 2008). Upon penetration of the host epidermis and establishment of the biotrophic interface 24 h after infection, expression of defense-related genes was suppressed, while a known cell-death suppressor, bax inhibitor 1 (BI-1), was transcriptionally induced (Eichmann et al., 2004; Doehlemann et al., 2008). In contrast to the situation during the compatible interaction, the suppression of defense responses is not observed after infection with an U. maydis mutant lacking the secreted effector protein Pep1 (protein essential during penetration 1) (Doehlemann et al., 2009). Pathogenic development of this mutant is blocked immediately upon epidermal penetration and elicits massive defense gene expression, accompanied by H2O2 accumulation and collapse of infected maize cells (Doehlemann et al., 2009), supporting the idea that Pep1 acts as a cell-death suppressor.

To elucidate which of the differentially regulated genes in these two situations actually determine compatibility or resistance in the maize–U. maydis interaction, efficient silencing of the respective maize genes is required. However, in monocot plants, and particularly in maize, production of stable RNA interference (RNAi) plants is not an appropriate method for the functional screening of a large number of candidate genes: regeneration of primary transformants takes c. 3–4 months and subsequent propagation and molecular characterization of individual lines is a time consuming and laborious procedure requiring large glasshouse capacities. Therefore, an alternative system that allows rapid and systemic gene silencing in maize is needed to facilitate the functional analysis of the numerous candidate genes derived from transcriptome analyses.

In addition to generation of transgenic plants, transient expression by particle bombardment or Agrobacterium tumefaciens-mediated gene transfer has been used in other systems, particularly in dicot plants such as Nicotiana benthamiana (Klein et al., 1988; Yang et al., 2000). For transient silencing of plant genes via RNAi, virus-induced gene silencing (VIGS) has become an important tool in recent years (Purkayastha & Dasgupta, 2009). Efficient VIGS systems have been established primarily in dicot plants such as N. benthamiana, where successful VIGS using a tobacco mosaic virus system was demonstrated in 1995 (Kumagai et al., 1995). Similarly, in Arabidopsis thaliana and legume systems, VIGS has been established based on cabbage leaf curl virus and pea early browning virus systems, respectively (Turnage et al., 2002; Constantin et al., 2004). Advanced systems allowing high-throughput silencing of tobacco genes have been developed using potato virus X (Lu et al., 2003) and the tobacco rattle virus (Chakravarthy et al., 2010). Moreover, A. tumefaciens has been successfully used to introduce viral DNA for induction of VIGS (Ratcliff et al., 2001; Liu et al., 2002). However, transient A. tumefaciens transformation of maize leaves has not been reported to date. In monocots, some examples of efficient VIGS have been reported. The most prominent system is based on the barley stripe mosaic virus (BSMV), which allows transient gene silencing in barley (Hordeum vulgare) and wheat (Triticum aestivum) (Holzberg et al., 2002; Bruun-Rasmussen et al., 2007). The BSMV system has been used to demonstrate a functional requirement for the wheat Lr21 resistance gene for leaf rust resistance (Scofield et al., 2005), to show the involvement of receptor-like kinases in wheat stripe rust resistance (Zhou et al., 2007), to functionally characterize the genes involved in barley Mla13-mediated resistance (Hein et al., 2005), and to show that the barley disease resistance gene Hm1 confers resistance to Cochliobolus carbonum race 1 (Sindhu et al., 2008). For maize, the brome mosaic virus (BMV) has been shown to be potentially suitable for VIGS. Using a modified BMV vector system based on cDNAs for BMV RNA1, RNA2 and RNA3, Ding et al. (2006) demonstrated silencing of the phytoene desaturase (PDS)-encoding gene in wheat, barley, rice (Oryza sativa) and maize. Ding and co-workers followed this up by optimizing BMV-mediated VIGS in rice (X. S. Ding & R. S. Nelson, pers. comm.).

For maize, however, a VIGS system that allows efficient and systemic gene silencing during interactions with fungal pathogens has not been developed to date. We now describe such a screening system for the maize–U. maydis interaction. Specifically, we optimized the BMV system previously described by Ding et al. (2006) and adapted it to experimental conditions that allow subsequent infection with the fungal biotroph U. maydis. A quantitative real-time PCR (qRT-PCR)-based test system allowed us to establish the roles of two maize genes in the interaction of U. maydis with maize.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant growth conditions and U. maydis infections

For VIGS experiments, Zea mays L. cv Va35 and ZmTIP1-YFP (Doehlemann et al., 2009; Mohanty et al., 2009) plants were grown in phytochambers at 28°C during the light period (26 000 lux; 14.5 h) and at 22°C during the dark period (9.5 h). Nicotiana benthamiana plants were grown at 22°C during the light period (26 000 lux; 14.5 h) and at 20°C during the dark period (9.5 h). For infections with U. maydis, liquid cultures of strain SG200 (Kämper et al., 2006) and SG200 Potef::2xRFP (Fuchs et al., 2006) were grown in YEPSL (0.4% yeast extract, 0.4% peptone and 2% sucrose), with shaking at 200 rounds min−1 (rpm), to an optical density (OD600) of 0.6–0.8. Cells were centrifuged at 900 g for 5 min, resuspended in H2O to an OD600 of 1 and used for infection of 17-d-old maize seedlings (11 d after BMV inoculation).

Plasmids

Standard molecular biology methods were used according to Sambrook et al. (1989).

pANDA/TPS  To silence terpene synthase 6/11 (tps6/11) (accession of full-length coding sequence: NM001112204) in maize by RNAi, the maize cDNA fragment was amplified by PCR (Supporting Information Table S1), cloned into the pENTR/D-TOPO vector (Invitrogen), and then transferred to the binary vector pANDA-b (provided by K. Shimamoto, Nara Institute of Science and Techn-ology, Ikoma, Japan) using the LR clonase™ reaction (Invitrogen, Carlsbad, California, USA).

pB3-3 constructs (pB3-3/TPSsi, pB3-3/ECBsi, pB3-3/BTIsi, pB3-3/BI-1si and pB3-3/YFPsi)  Primers were designed for tps6/11 (full-length coding sequence: NM001112204), endochitinase B (ecb) (NM001156000), Bowmann–Birk type trypsin inhibitor (bti) (NM001153399) and bax inhibitor 1 (bi-1) (AY105656). All primers contained a HindIII restriction site for integration into pB3-3 (Table S1). PCR fragments for induction of VIGS were amplified from cDNA of U. maydis-infected Va35 plants and were tested by sequencing before cloning into pB3-3. For construction of pB3-3/YFPsi, a fragment of yfp (yellow fluorescence protein) was amplified from p123-yfp (Table S1; Weber et al., 2003). All PCR fragments were digested with HindIII and ligated into the HindIII site of pB3-3 (Ding et al., 2006). The following maize gene fragments were inserted in antisense orientation to RNA3: for tps6/11, 166 bp; for ecb, 181 bp; for bti, 187 bp; for bi-1, 152 bp; and for yfp, 171 bp. For in silico analysis of siRNA formation and the silencing specificity of maize sequences, the software tool siRNA Scan (http://bioinfo2.noble.org/RNAiScan.htm) was used. Predictions were made using data from the J. Craig Venter Institute maize tgi v16 database. pF1-11 and pF2-2 were provided by X. S. Ding & R. S. Nelson (Ding et al., 2006).

In vitro transcription

To obtain BMV RNA1, RNA2 and RNA3, the plasmid pF1-11, pF2-2 and the different pB3-3 constructs were individually digested in 50-μl reactions containing 3 μg of template DNA and 1.5 μl of SpeI or PshAI NEB (New England Biolabs, Frankfurt am Main, Germany) for 1.5 h at 37°C. Afterwards, the restriction enzymes were heat-inactivated and the sample volume was reduced to a final volume of 2 μl. Individual transcripts were synthesized using the mMessage mMachine Kit (Ambion). Before plant inoculation, the synthesized RNA was tested using agarose gel electrophoresis.

BMV infection

Nicotiana benthamiana plants were infected as described by Ding et al. (2007). Seven days after inoculation, the leaves were harvested and ground in 0.1 M phosphate puffer, pH 6.0 (1 : 10, w/v). The BMV titer was quantified by qPCR using primers specific for the minus strand of RNA1 (Table S1). All N. benthamiana extracts were adjusted by addition of 0.1 M phosphate puffer, pH 6.0, to the same virus titer of 10 000 relative expression units compared with noninoculated tobacco. For maize infection, the second leaf of 6-d-old maize plants was dusted with carborundum (400 mesh; Sigma) and 50 μl of N. benthamiana extract was gently rubbed onto the leaf surface.

cDNA synthesis and qRT-PCR

To determine the fungal biomass as well as expression levels of the RNAi target genes in maize leaves, 2–5-cm sections of the infected leaves were excised 1 cm below the U. maydis injection site. In the cases of bi-1, bti and ecb, U. maydis-infected samples were taken 48 h after fungal infection (13 d after BMV inoculation) to determine gene expression and fungal proliferation. For tps6/11, samples for qRT-PCR were taken 8 d after U. maydis infection from VIGS experiments and stable RNAi lines, respectively. To determine pds-silencing efficiency in different maize leaves, samples were taken 5 d (for leaf 4), 7 d (for leaf 5) and 11 d (for leaf 6) after BMV/PDSsi (BMV containing a sequence for phytoene desaturase silencing) inoculation. To remove fungal cells from the leaf surface, the sections were washed three times with 0.1% Tween 20 in water. For subsequent gDNA and RNA extraction, each leaf was frozen in liquid nitrogen and ground to powder and extracted using the MasterPure Complete DNA & RNA Purification Kit (Biozym, Oldendorf, Germany). After extraction, the First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) was used to reverse-transcribe 1 μg of total RNA with oligo(dT) primers for qRT-PCR. For the insert stability test for RNA3, 1.8 μg of total RNA was reverse-transcribed using random hexamer primers. The qRT-PCR analysis was performed using an iCycler machine (Bio-Rad) in combination with iQ SYBR Green Supermix (Bio-Rad). For virus titer detection, 2 μl of tobacco extracts was used for qPCR after centrifugation at 13 000 g for 2 min. For detection of BMV titers, primers binding the minus strand of BMV RNA1 (Table S1) were used for qPCR. Minus-strand RNA1 was calculated relative to the amount of tobacco actin (AY594294; Table S1). Primers for quantification of U. maydis biomass, maize gene transcription levels and virus titer detection are summarized in Table S1. Maize glyceraldehyde dehydrogenase (gapdh; NM001111943) was used as a standard gene for normalization. Cycling conditions were as follows: 2 min at 95°C, followed by 45 cycles of 30 s at 95°C, 30 s at 61°C and 30 s at 72°C. After each PCR, the specificity of the amplified product was verified and the threshold cycle above background was calculated using Bio-Rad iCycler software. Gene expression levels and the relative amount of fungal DNA were then calculated relative to gapdh expression levels or the amount of gapdh DNA, respectively. Relative quantification of gene expression and calculation of fungal DNA were carried out using a relative expression software tool (REST©, Qiagen, Hilden, Germany), applying an improved ΔΔ analysis (Pfaffl et al., 2002). Error bars in all figures that show qRT-PCR data give the standard deviation that was calculated from the original CT (cycle threshold) values. For each individual experiment, five BMV/YFPsi control plants were analyzed to calculate mean values that were set to 100% relative to material from BMV-silenced samples. P-values were estimated using hypothesis test P(H1).

Maize transformation

Transgenic maize plants were produced by A. tumefaciens-mediated gene transfer largely following the protocol of Hensel et al. (2009). Donor plants (Hi II A × Hi II B) were selfed and immature embryos of F2 hybrid plants (Hi II (A × B) × (A × B)) were transformed. In the present study, A. tumefaciens strain AGL-1 (Lazo et al., 1991) was used, the co-culture medium contained 500 μM acetosyringone and the concentration of the selective agent Bialaphos (Molekula, Taufkirchen, Germany) was 2 mg l−1 in the first and 4 mg l−1 in the second selection step.

Confocal microscopy of infected plant tissue

YFP fluorescence in ZmTIP1-YFP plants, expressing an aquaporin localizing to the tonoplast membrane fused to yfp, was monitored in BMV/YFP-inoculated fresh maize tissue using excitation with a 488-nm laser and detection at 510–550 nm. For red fluorescent protein (RFP) fluorescence of SG200 Potef::2xRFP hyphae in maize tissue, an excitation of 561 nm and detection at 580–630 nm were used. Confocal images were taken on a TCS-SP5 confocal microscope (Leica, Wetzlar, Germany).

Western blot

For detection of BMV in maize leaves, immunodetection of the BMV coat protein was performed. Mock-treated material and maize leaves that were inoculated with BMV containing a sequence for phytoene desaturase silencing (BMV/PDSsi) were collected 6 d (for leaf 4), 8 d (for leaf 5) and 12 d (for leaf 6) after inoculation and ground in liquid nitrogen. Extraction buffer (50 mM HEPES, pH 7.5; 2 mM Dithiothreitol; 0.1% sodium dodecyl sulphate (SDS); 0.01% bovine serum albumin (BSA)) was then added and incubated for 30 min, and insoluble material was removed by repeated centrifugation. Thirty micrograms of protein was subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and subsequently blotted on nitrocellulose membrane. After blocking with 5% nonfat milk, the anti-BMV capture antibody (Agdia, Elkhart, Indiana, USA) was incubated overnight in a 1 : 100 dilution. The second antiserum anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase (Cell Signaling, Danvers, Massachusetts, USA) was applied in a 1 : 7500 dilution. For signal detection, the chemiluminescent substrate femto ECL (Pierce, Rockford, Illinois, USA) was used.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Infection of maize with BMV and U. maydis requires strictly defined experimental conditions

As a prerequisite for the identification of maize genes involved in the maize–U. maydis interaction, the VIGS system had to be adapted to experimental conditions that allowed U. maydis infection of maize plants that had been preinfected with BMV. Critical parameters for both BMV and U. maydis are temperature, light intensity and inoculum quantity. While U. maydis prefers temperatures c. 30°C ± 5°C for efficient infection, in our hands BMV appeared to be sensitive to high temperatures, in particular at the time of inoculation (data not shown). Therefore, BMV infections were carried out at the beginning of the night period where the absence of light and a temperature of 22°C promoted efficient viral infection. Fungal infections were performed at the beginning of the 14-h light period, when the temperature was set to 28°C to support fungal development (see the Materials and Methods section for details).

In addition to defined growth conditions, standardization of the BMV infection system required a constant virus titer in the inocula for all independent experiments. To this end, in vitro transcribed BMV RNA was initially inoculated into N. benthamiana, which is highly susceptible to BMV and therefore an adequate intermediate host (Ding et al., 2007). Six days after the inoculation of two leaves of N. benthamiana plants, mosaic symptoms were locally visible on the infected leaf as well as on the upper leaves of infected plants, indicating systemic infection (not shown). At this stage, leaf extracts of the BMV-infected N. benthamiana plants were prepared and analyzed by qPCR to quantify BMV RNA1. To guarantee comparable inoculum quantity in all VIGS experiments described below, virus titers of all constructs were adjusted to a constant level (see the Materials and Methods section for details).

BMV-mediated gene silencing is systemic and does not interfere with U. maydis infection

While BMV can multiply in mature leaves, U. maydis only infects primordial tissue exhibiting mitotic activity (Wenzler & Meins, 1987). Therefore, a systemic spread of BMV needs to be established before U. maydis infection to facilitate VIGS in plant cells colonized by fungal hyphae. As a standard, 6-d-old maize seedlings were inoculated with BMV at the two-leaf stage. Five to six days after inoculation, systemic spread of virus-induced mosaic symptoms became evident in leaf 4 of infected maize seedlings (data not shown). Using an antibody specific to the BMV coat protein, systemic viral infection was confirmed in maize leaves 4 to 6, at 12, 14 and 18 d after inoculation (Fig. S1). In a previous study, photo-bleaching as a result of BMV-mediated gene silencing of the maize pds gene was reported to be visible as early as 10 d after inoculation (Ding et al., 2006). To identify the best time-point and site of infection for U. maydis, the efficiency of BMV-induced VIGS was determined in different maize leaves. Similar to the observations made by Ding et al. (2006), inoculation of maize with BMV containing a sequence for silencing of pds (BMV/PDSsi) induced photo-bleaching in the upper leaves. However, the efficiency and reproducibility of VIGS appeared to depend on the tested leaf: the strongest phenotype was observed in leaf 6, where photo-bleaching was evident and reproducible in all plants tested. In leaves 4 and 5, similar symptoms were observed, but photo-bleaching appeared to be milder and was not observed in all tested plants (Fig. 1a and not shown). For a precise quantification of the silencing efficiency in different leaves, mRNA of BMV/PDSsi infected plants was prepared from leaves 4 to 6 and transcript levels of pds were determined using qRT-PCR. Leaf 6 showed a reproducible, efficient silencing of 24–81% compared with control plants, correlating with the occurrence of photo-bleaching (Fig. 1b). Silencing of pds in leaves 4 and 5 was less effective and, most importantly, highly variable in individual plants (Fig. 1b). Based on these findings, the following infection procedure was defined. BMV was inoculated into the second leaf of 6-d-old maize seedlings. Subsequently, 11 d after BMV inoculation, the sixth leaf of the maize plants was infected with U. maydis.

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Figure 1.  Efficiency of brome mosaic virus (BMV)-mediated gene silencing in different maize leaves. (a) BMV/PDSsi (BMV containing a sequence for phytoene desaturase silencing) was inoculated into the second leaf of 6-d-old maize seedlings; 11 d after BMV inoculation, systemic silencing of phytoene desaturase (pds) resulted in visible photo-bleaching of upper leaves 5 and 6. (b) Quantitative real-time (qRT)-PCR quantification of pds silencing in eight individual plants 5 d (for leaf 4), 7 d (for leaf 5) and 11 d (for leaf 6) after BMV inoculation demonstrated the high variability of silencing efficiency in leaves 4 and 5, while efficient silencing was consistently observed in leaf 6. Error bars represent SD. *, P < 0.1; **, P < 0.05.

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To visualize systemic, BMV-mediated gene silencing at the cellular level, maize plants constitutively expressing a YFP-tagged TIP-1 (tonoplast intrinsic protein 1) fusion protein (Doehlemann et al., 2009; Mohanty et al., 2009) were inoculated with BMV/YFPsi. Fluorescence microscopy of systemically infected leaves showed efficient silencing of YFP fluorescence in large cell-clusters, mainly in the vicinity of vascular bundles (Fig. 2a). A central issue was to verify that BMV infection did not interfere with the virulence of U. maydis. To address this point, maize plants showing systemic virus symptoms were infected with the solopathogenic U. maydis strain SG200 (Kämper et al., 2006). Scoring of U. maydis-induced tumor formation 12 d after SG200 infection demonstrated that disease development was not affected by prior BMV inoculation (Fig. 2b). Furthermore, confocal microscopy of rfp-expressing U. maydis hyphae in BMV/YFPsi-infected tip1-yfp-expressing plants showed that U. maydis infection did not interfere with VIGS, while U. maydis could penetrate yfp-silencing cells (Fig. 2c).

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Figure 2.  Brome mosaic virus (BMV)-induced virus-induced gene silencing (VIGS) of yfp (yellow fluorescence protein) does not interfere with Ustilago maydis infection. (a) Left panel, control. Middle panel, epifluorescence image of a maize leaf section expressing tip1-yfp (tonoplast intrinsic protein 1-yellow fluorescence protein) (Doehlemann et al., 2009; Mohanty et al., 2009) showing strong, constitutive YFP fluorescence in all leaf cells around a vascular bundle (asterisk). Right panel, epifluorescence images of tip1-yfp-expressing maize leaves that were infected with BMV/YFPsi (BMV containing a sequence for yellow fluorescence protein silencing). Here, 11 d after inoculation, large clusters of maize cells were silenced for yfp expression. This phenotype was observed predominantly in the proximity of vascular tissue (asterisks). (b) Seventeen-day-old maize plants of cv Va35 were infected with SG200 11 d after BMV or mock inoculation. Ustilago maydis symptoms were scored 8 d after fungal infection in four independent experiments. Ustilago maydis disease symptoms of BMV- and mock-inoculated plants were not significantly different (Welch’s unpaired t-test, P = 0.362). Error bars represent SD. (c) Confocal projections of tip1-yfp-expressing maize leaves that were infected with an U. maydis strain expressing cytoplasmic rfp (Fuchs et al., 2006). Left panel: 48 h after U. maydis infection, biotrophic fungal hyphae (white arrowheads) grow inside the YFP-expressing maize cells. Tip1-YFP fluorescence surrounds plant nuclei (n) and vacuoles (v) constitutively in all cells. Right panel: intracellular U. maydis hyphae (white arrowheads) 48 h after fungal infection of plants where YFP has been silenced by BMV/YFPsi. Biotrophic hyhae grow in cells (silhouettes marked by dashed lines) that do not show TIP-YFP signals.

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To evaluate the influence of gene silencing on the interaction of maize with U. maydis, the amount of in planta fungal DNA was determined by qPCR. As a control, U. maydis colonization was quantified in leaves of plants that were inoculated with BMV/YFPsi. It was expected that silencing of maize genes with a functional role during interaction with U. maydis would result in reduced or increased fungal colonization. To obtain a direct correlation of VIGS efficiency and U. maydis virulence, each plant was assayed individually. BMV- and U. maydis-infected leaf material of individual plants was collected and used for isolation of genomic DNA and total RNA, allowing quantification of both VIGS efficiency and the amount of fungal material in the same qRT-PCR experiment (Fig. 3).

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Figure 3.  Experimental set-up for a quantitative readout to investigate the influence of systemic virus-induced gene silencing (VIGS) in maize on the interaction with Ustilago maydis. GOI, gene of interest. For details, see text.

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VIGS allows functional analysis of the maize–U. maydis interaction

To test the screening system described in this study, a set of maize genes was selected for further analysis. Candidate genes were selected based on previous expression profiling of maize during interaction with the U. maydis strain SG200 and the nonpathogenic mutant SG200 Δpep1 (Doehlemann et al., 2008, 2009). A gene encoding for TPS6/11 (Basse, 2005; Köllner et al., 2008) was found to be the most strongly up-regulated maize gene after U. maydis infection, both in the pre-penetration phase (12 hours post infection) and after establishment of biotrophy (24 hpi). Strong tps6/11 induction was also detected in SG200Δpep1-infected leaves 24 h after infection, a time-point at which growth of the mutant is blocked by the plant defense response (Table 1; Doehlemann et al., 2009). Furthermore, a gene encoding an ECB (CD967190) was found to be strongly up-regulated 12 h after infection with SG200 (Table 1). During the early biotrophic interaction as well as during the incompatible interaction with SG200Δpep1, this gene remained induced; however, its expression was significantly lower when compared with the 12-h time-point. The third candidate gene, encoding for BTI (BM380261), was found to be specifically induced > 200-fold at 24 h after SG200 infection compared with mock-infected leaves (Table 1). Last, BI-1 was by VIGS, a highly conserved cell-death inhibiting protein, which has previously been shown to be functionally involved in the biotrophic interaction of barley with Blumeria graminis f.sp. hordei (Eichmann et al., 2004).

Table 1.   Maize genes tested by virus-induced gene silencing (VIGS) and their role during Ustilago maydis infection
GeneGenBank accessionFC1 SG200 12 hpiFC1 SG200 24 hpiFC1Δpep1 24 hpi
  1. 1FC, fold change of transcriptional induction compared with mock-infected maize leaves at the same time-point. Expression data were taken from Doehlemann et al. (2008, 2009).

  2. 2ns, no significant change in gene expression.

Terpene synthase 6/11 (tps6/11)CF014750725.53x349.59x1781.73x
Endochitinase B (ecb)CD967190456.57x 32.46x  94.81x
Bowmann–Birk type trypsin inhibitor (bti)BM380261ns2210.65xns2
Bax inhibitor 1 (bi-1)CN844282  3.32x  1.31x   3.96x

Inoculation of BMV/ECBsi and BMV/BTIsi resulted in significant reduction in transcript levels for both maize genes (Fig. S2). In one out of five bti-silenced plants, fungal colonization was significantly reduced compared with BMV/YFPsi-inoculated controls. In the other four plants, however, no significant effect was observed. Consequently, calculation of the mean values for all tested plants did not show a significant effect of bti silencing on U. maydis infection (Fig. S2a). In the case of ecb, all five plants with significant silencing of the ecb gene showed altered fungal colonization in comparison with the control samples (Fig. S2b). While two plants showed slightly reduced colonization, three other plants contained increased amounts of U. maydis DNA. As a result of these heterogeneous data, calculation of the mean values for all tested plants showed no significant effect of ecb silencing on U. maydis colonization (Fig. S2b). Taken together, these findings indicate that neither bti nor ecb, despite their transcriptional activation, has a detectable impact on the maize–U. maydis interaction.

By contrast, a functional role during biotrophic interaction became evident for BI-1: VIGS of bi-1 resulted in reduced maize plant size; however, apart from this growth phenotype, plants did not show any morphological alterations (Fig. 4a). When infected with U. maydis, bi-1-silenced plants displayed dark, necrotic lesions at sites of fungal infection and no tumor formation was observed 8 d after fungal infection (Fig. 4b). Moreover, in four out of five plants silenced for bi-1, qPCR revealed a significant reduction in U. maydis colonization as early as 48 h after fungal infection (Fig. 4c). Calculation of mean values for all tested plants confirmed a significant reduction in fungal colonization in the bi-1-silenced plants by c. 40% (Fig. 4c). In order to confirm that these effects were a direct result of BMV-mediated gene silencing, the insert stability of the BMV/BI-1 construct was tested in bi-1-silenced maize plants. cDNA of respective leaves was prepared and used for PCR with primers specific for BMV RNA3 containing the inserted bi-1 fragment. The expected 199-bp PCR product, originating specifically from the silencing construct, was amplified from two independent maize plants showing bi-1 silencing as well as from BMV/BI-1si-infected N. benthamiana, but not from a mock-infected control sample (Fig. S3).

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Figure 4.  Systemic virus-induced gene silencing (VIGS) of maize bax inhibitor 1 (bi-1) causes stunted plant growth but increases resistance to Ustilago maydis. (a) Brome mosaic virus (BMV)/BI-1si (BMV containing a sequence for Bax Inhibitor-1 silencing) infection resulted in stunted growth of maize plants compared with BMV/YFPsi (BMV containing a sequence for yellow fluorescence protein silencing) controls. (b) In BMV/BI-1si-infected plants, U. maydis infection resulted in formation of necrotic lesions along the leaf blade, as indicated by arrowheads. Pictures were taken 8 d after infection (dai) with U. maydis strain SG200. (c) Fungal DNA and bi-1 expression were quantified by quantitative real-time (qRT)-PCR using leaf samples from bi-1-silenced plants and BMV/YFPsi-infected control plants 48 h after U. maydis infection. The relative amount of U. maydis DNA (white columns) and the relative expression of bi-1 (black columns) in five BMV/YFPsi control plants were averaged and set to 100% (mean BMV/YFPsi). Columns BMV/BI-1si 1–5 show fungal DNA and bi-1 expression of five individual silenced plants; the column mean BMV/BI-1si gives the average values for these bi-1-silenced plants. Error bars represent the standard deviation. *, P < 0.1; **, P < 0.05.

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A functional role during the maize–U. maydis interaction was also evident for tps6/11. Inoculation of maize seedlings with BMV/TPSsi did not cause any visible effects on plant growth and morphology compared with BMV/YPFsi-treated control plants (not shown). Interestingly, silencing of tps6/11 resulted in a significant increase in susceptibility to U. maydis. qPCR quantification showed increased levels of U. maydis DNA in all plants in which there was silencing of tps6/11 (Fig. 5a). In addition, tps6/11-silenced plants exhibited faster symptom development and extensive chlorosis upon U. maydis infection (Fig. 5b and not shown). From these results we conclude that the dramatic transcriptional induction of tps6/11, which was observed in wild-type plants (Doehlemann et al., 2008), is required to restrict progression of U. maydis disease development.

image

Figure 5.  Systemic virus-induced gene silencing (VIGS) of maize terpene synthase 6/11 (tps6/11) results in increased colonization of maize leaves by Ustilago maydis. (a) Quantitative real-time (qRT)-PCR of fungal DNA and tps6/11 transcript levels from the same leaf material 8 d after U. maydis infection (dai) shows increased fungal colonization in leaves where tps6/11 has been silenced. The relative amount of U. maydis DNA (white columns) and the relative expression of tps6/11 (black columns) in five brome mosaic virus BMV/YFPsi (BMV containing a sequence for yellow fluorescence protein silencing)-infected control plants (mean BMV/YFPsi) were averaged and set to 100%. Columns BMV/TPSsi 1–5 show fungal DNA and bax inhibitor 1 (bi-1) expression of five individual silenced plants. The column mean BMV/TPSsi gives the average values for these tps6/11-silenced plants. Error bars represent SD. *, P < 0.1; **, P < 0.05. (b) Disease symptoms caused by U. maydis strain SG200 in tps6/11-silenced maize leaves (BMV/TPSsi) compared with a typical BMV/YFPsi-infected control leaf of maize line Va35. Images were taken 8 d after fungal infection.

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Stable RNAi lines for tps6/11 verify the VIGS results

Although BMV infection itself had no obvious influence on the pathogenic development of U. maydis (Fig. 1), the possibility of unexpected effects of viral infection attributable to VIGS could not be entirely excluded. To investigate this possibility, maize lines carrying a stable integration of an RNAi construct for tps6/11 were produced. Two inverted repeats of a 428-bp fragment of the tps6/11 coding region were introduced into plasmid pANDA-b to express double-stranded mRNA under control of the maize ubiquitin promoter. After A. tumefaciens-mediated stable transformation of maize, 14 independent T0 lines were obtained that had been tested for successful integration of the RNAi construct (not shown). The stably transformed plants did not show any morphological differences compared with nonsilenced plants with the same genotype (Hi II (A × B) × (A × B)) (Fig. 6c). Similar to the experimental procedure for the VIGS experiments described for TPS6/11, the sixth leaf of the transgenic plants was infected with U. maydis. Plants were scored for U. maydis symptoms, the efficiency of tps6/11 silencing and the amount of fungal biomass in the infected tissue 8 d after fungal infection. Of the 14 independent transgenic plants that were infected with U. maydis, 10 did not show silencing of tps6/11 (not shown). Two plants (tpsRNAi11 and tpsRNAi25) showed weak silencing with an efficiency of < 50%, and in these plants U. maydis colonization was not significantly different from that in nonsilenced controls (Fig. 6a). In two of the U. maydis-infected plants (tpsRNAi18 and tpsRNAi24), tps6/11 expression was silenced efficiently and 8 d after fungal infection these plants contained significantly increased levels of U. maydis colonization (Fig. 6a). This was associated with increased tumor formation and extended chlorosis (Fig. 6b), similar to the observations made for the plants silenced with BMV/TPSsi (Fig. 5). These results confirm the data obtained from the VIGS experiments and demonstrate the successful application of BMV-mediated gene silencing for the functional analysis of the maize–U. maydis interaction.

image

Figure 6.  Stable RNA interference (RNAi) of terpene synthase 6/11 (tps6/11) increases susceptibility to Ustilago maydis. Transgenic plants are termed tpsRNAi. Numbers designate individual T0 plants, representing individual transformation events. (a) Quantitative real-time (qRT)-PCR was performed to quantify U. maydis DNA (white columns) and tps6/11 transcript levels (black columns). Leaf material was taken 8 d after U. maydis infection (dai). Compared with nonsilenced control plants (mean wild type (WT)), significantly increased amounts of U. maydis DNA were found in maize lines tpsRNAi18 and tpsRNAi24, which also showed efficient RNAi of tps6/11. In transgenic lines tpsRNAi11 and tpsRNAi25, silencing of tps6/11 was less efficient, and in these plants U. maydis colonization was comparable to that of control plants (mean WT). The relative amount of U. maydis DNA and tps6/11 expression in two U. maydis-infected control plants (mean WT) was averaged and set to 100%. Error bars represent SD. *, P < 0.1; **, P < 0.05. (b) Ustilago maydis symptoms on tps6/11 RNAi T0 lines and WT plants 8 d after fungal infection. Transgenic plants tpsRNAi18 and tpsRNAi24 showed bigger tumors and stronger chlorosis than WT and transgenic plants tpsRNAi11 and tpsRNAi25. (c) Noninfected leaves of tps6/11 RNAi plants were indistinguishable from those of WT plants of the same crossing (Hi II (A × B) × (A × B)).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Functional analyses in monocotyledonous pathosystems require transient approaches

In various dicot plants, particularly in the model organism A. thaliana, receptor proteins for pathogen elicitors and signaling cascades, which are key players in the plant immune system, have been identified (Nishimura & Dangl, 2010). In monocot plants, the interaction between barley plants and the powdery mildew fungus B. graminis f. sp. hordei has been studied intensively to identify the mechanisms that facilitate accommodation of biotrophic fungi (Eichmann & Hückelhoven, 2008). For maize, however, there is only fragmentary information about the mechanisms of resistance to, or compatibility with, fungal pathogens. While several virulence factors of U. maydis have previously been identified (Kämper et al., 2006; Brefort et al., 2009), there is still little information about maize genes that determine this interaction; for example, there is no maize resistance gene known for U. maydis. Recently, microarray-based transcriptome profiling approaches to the study of various pathogen interactions in monocot plants have identified a large number of plant genes that show differential regulation during infection (Caldo et al., 2004; Wise et al., 2007; Doehlemann et al., 2008). These studies are largely descriptive and leave the most intriguing question unanswered: which of the genes being regulated in response to pathogen infection actually possess a functional role in the interaction? To enable functional characterization of the long lists of candidate genes, a VIGS-based screening system that allows systemic silencing during pathogen interaction has been successfully established for maize.

Experimental set-up of the quantitative test system

It was instrumental to this study to identify experimental conditions that support pathogenic development of both BMV and U. maydis. Previously, it was reported that alteration of plasmodesmata causes increased spreading of BMV in the mesophyll at higher temperatures, while mainly vascular bundles are infected at lower temperatures (Ding et al., 1999). In our hands, inoculation of BMV required a low temperature, while a high temperature during the day was applied to favor U. maydis infection. Consequently, under these conditions, silencing of yfp was mainly observed in the mesophyll, while the vascular bundles remained fluorescent. Furthermore, the finding that BMV infection before U. maydis infection did not interfere with the pathogenic development of the fungus provided the basis for the functional screening system. This is of particular interest, as it was previously reported that in various BSMV–barley interactions viral disease symptoms interfered with the loss-of-function phenotype caused by silencing of the target gene (Hein et al., 2005; Scofield et al., 2005). BMV infection was reported to cause local cell death in barley and for certain maize genotpyes, such as B73, systemic necrosis in response to BMV infection has been reported (Ding et al., 2001). For maize cv Va35, which was used in this study, Diaminobenzidine (DAB) and trypan blue staining did not show BMV-induced cell death (Ding et al., 2001). In addition, introduction of a nonvirus gene fragment into RNA3 caused a reduction in virulence, leading to reduced BMV disease symptoms (data not shown). For this reason, control experiments for silencing approaches were performed with a VIGS construct containing a nonplant gene fragment (yfp), a practice that has also been suggested by Scofield & Nelson (2009). The defined virus titer and experimental conditions applied for all experiments in this study resulted in mild virus symptoms but allowed systemic silencing. Interestingly, the efficiency and, in particular, the reproducibility of VIGS depended on the tested maize leaf. While silencing could be observed in leaves 4 and 5, its efficiency was highly variable between individual plants. Only in leaf 6 VIGS was found to be highly reproducible. In later developing leaves, however, silencing was found to be dramatically reduced, indicating the transient character of VIGS and the high degree of resistance to BMV in maize cv Va35. Despite the high reproducibility of VIGS in leaf 6, assessment of the functional roles of maize genes during the U. maydis interaction essentially requires quantitative evaluation of individual plants. Only the direct correlation between VIGS efficiency and the amount of fungal biomass in individual samples guarantees a reliable evaluation of gene function.

Careful selection of sequences for insertion into BMV RNA3 is also critical for efficient and specific gene silencing. In this study, insert sequences were tested in silico using the software tool siRNA Scan (http://bioinfo2.noble.org/RNAiScan.htm) to predict the number of siRNAs formed from selected sequences. Nevertheless, depending on the target gene, silencing efficiency was variable, with an average of c. 70% for bti but only c. 50% for ecb.

RNAi in plants is not a cell-autonomous event; silencing can spread throughout the plant tissue, mediated by siRNAs that are translocated via plasmodesmata. Before responding to this signal, the receiving cells might require a critical siRNA dosage (Tournier et al., 2006). This model correlates with our microscopic observation in yfp-silenced TIP1-YFP maize plants, where silencing was shown to be highly efficient in individual cells. However, in addition to clusters of silenced cells, we also observed regions where cells showed full fluorescence, indicating a dose dependence of silencing. This mixture of silenced and nonsilenced cells is probably the cause of the rather moderate silencing efficiency measured in the qRT-PCR-based readout, where nucleic acids for PCR were extracted from whole leaf sections. For a functional analysis of maize genes during fungal interaction, highly efficient silencing in individual cells is likely to be more important than the overall intermediate effect on all plant cells: while a slight reduction in expression of a compatibility factor such as bi-1 might cause only marginal effects, its absence would stop fungal development in individual, colonized host cells and lead to a visible incompatibility reaction.

VIGS of pathogen-induced maize genes during U. maydis infection

For a functional test of the method described in this study, four U. maydis-induced maize genes were analyzed. The VIGS of ecb and bti did not cause significant changes after U. maydis infection, indicating minor roles of these proteins in this biotrophic interaction. In the case of bti, in particular, this was not surprising. Being induced by jasmonic acid during wounding responses, these BTI proteins are typically components of plant defenses against herbivore attack (Rohrmeier & Lehle, 1993; Rakwal et al., 2001). Induction of bti during U. maydis infection might be a consequence of the activation of jasmonic acid signaling during biotrophy (Glazebrook, 2005; Doehlemann et al., 2008) rather than a specific defense response following fungal infection.

The role of the cell-death suppressor BI-1 in the interaction with fungal pathogens has already been demonstrated in barley. Overexpression of barley bi-1 increased susceptibility to B. graminis f. sp. tritici by suppression of epidermal cell death upon fungal penetration, while necrotrophic invasion by Phakopsora pachyrhizi was reduced (Eichmann et al., 2004; Höfle et al., 2009). VIGS of maize bi-1 resulted in a reduction in the size of maize plants, but did not cause any further macroscopic symptoms. However, U. maydis infection was significantly reduced in these plants, and at sites of infection necrotic lesions were observed. This is consistent with the observations in barley and supports the idea that cell-death suppression is required to favor biotrophic pathogens and identifies BI-1 as a compatibility factor in the maize–U. maydis interaction.

For tps6/11, the opposite phenotype was observed, and silencing resulted in increased susceptibility of maize to U. maydis. TPSs are thought to act in defense responses to herbivore attack (Köllner et al., 2009) while their role in defense against pathogenic microbes is largely unknown. Nevertheless, a possible function of terpenoids as phytoalexins is discussed, because many of those compounds possess direct antimicrobial properties (Soković & Griensven, 2006). Alternatively, products of TPS might participate in defense signaling. It has been shown that the monoterpene allo-ocimene induces resistance and primes defense reactions against the fungal pathogen Botrytis cinerea (Kishimoto et al., 2006). The results obtained with the transient VIGS system have been successfully confirmed using stable RNAi lines for tps6/11. To date, production of T1/T2 generation plants is ongoing. These plants can be used in future studies to elaborate how sesquiterpenes resulting from TPS6/11 activity contribute to defense against U. maydis. To date there are no gene-for-gene relationships described for the maize–U. maydis pathosystem and tps6/11 is the first maize gene identified that is required to restrict U. maydis infection. In addition, the role of TPS6/11 during interaction with other pathogens will be investigated in future work.

Successful VIGS of bi-1 and detection of its impact on U. maydis pathogenicity will now allow the identification of maize genes that act systematically as compatibility factors. Moreover, the established system is a versatile tool to functionally study interactions with other fungal pathogens and therefore provides a significant technical improvement to the community.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to X. S. Ding and R. Nelson for providing the BMV vectors pF1-11, pF2-2, pB3-3 and pB3-3/PDS240 and for helpful advice and discussions about VIGS in maize. Va35 kernels were kindly provided by M. J. Millard, the Maize Genetics Cooperation Stock Center. For ZmTip1-YFP we thank D. Jackson. For providing pANDA-b we thank K. Shimamoto. The excellent technical assistance of H. Büchner and S. Wolf is gratefully acknowledged. For critical comments on the manuscript we thank A. Zuccaro, P. Berndt and S. Treitschke. The project was financed by the Deutsche Forschungsgemeinschaft (DFG) via research group FOR666 and IMPRS-Mic.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Western blot detection of the brome mosaic virus (BMV) coat protein in systemically infected maize leaves.

Fig. S2 Virus-induced gene silencing (VIGS) of maize endochitinase B (ecb) and Bowmann–Birk type trypsin inhibitor (bti) genes.

Fig. S3 PCR on brome mosaic virus (BMV) RNA3 with insertion of a bax inhibitor 1 (bi-1) fragment to demonstrate insert stability of silencing constructs in systemically infected maize leaves.

Table S1 List of PCR primers used in this study

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