Necrotrophic Fungus Sclerotinia Sclerotiorum Research Articles

Natural variation in BnaA07.MKK9 confers resistance to Sclerotinia stem rot in oilseed rape

Sclerotinia stem rot (SSR), caused by the necrotrophic fungus Sclerotinia sclerotiorum, is one of the most devastating diseases for several major oil-producing crops. Despite its impact, the genetic basis of SSR resistance in plants remains poorly understood. Here, through a genome-wide association study, we identify a key gene, BnaA07. MKK9, that encodes a mitogen-activated protein kinase kinase that confers SSR resistance in oilseed rape. Our functional analyses reveal that BnaA07.MKK9 interacts with BnaC03.MPK3 and BnaC03.MPK6 and phosphorylates them at the TEY activation motif, triggering a signaling cascade that initiates biosynthesis of ethylene, camalexin, and indole glucosinolates, and promotes accumulation of H2O2 and the hypersensitive response, ultimately conferring resistance. Furthermore, variations in the coding sequence of BnaA07.MKK9 alter its kinase activity and improve SSR resistance by ~30% in cultivars carrying the advantageous haplotype. These findings enhance our understanding of SSR resistance and may help engineer novel diversity for future breeding of oilseed rape.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against thebroad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

Non-proteinogenic amino acids mitigate oxidative stress and enhance the resistance of common bean plants against Sclerotinia sclerotiorum.

White mold, caused by the necrotrophic fungus Sclerotinia sclerotiorum, is a challenging disease to common bean cultivation worldwide. In the current study, two non-proteinogenic amino acids (NPAAs), γ-aminobutyric acid (GABA) and ß-alanine, were suggested as innovative environmentally acceptable alternatives for more sustainable management of white mold disease. In vitro, GABA and ß-alanine individually demonstrated potent dose-dependent fungistatic activity and effectively impeded the radial growth and development of S. sclerotiorum mycelium. Moreover, the application of GABA or ß-alanine as a seed treatment followed by three root drench applications efficiently decreased the disease severity, stimulated plant growth, and boosted the content of photosynthetic pigments of treated S. sclerotiorum-infected plants. Furthermore, although higher levels of hydrogen peroxide (H2O2), superoxide anion (O2 •-), and malondialdehyde (MDA) indicated that S. sclerotiorum infection had markedly triggered oxidative stress in infected bean plants, the exogenous application of both NPAAs significantly reduced the levels of the three studied oxidative stress indicators. Additionally, the application of GABA and ß-alanine increased the levels of both non-enzymatic (total soluble phenolics and flavonoids), as well as enzymatic (catalase [CAT], peroxidases [POX], and polyphenol oxidase [PPO]) antioxidants in the leaves of S. sclerotiorum-infected plants and improved their scavenging activity and antioxidant efficiency. Applications of GABA and ß-alanine also raised the proline and total amino acid content of infected bean plants. Lastly, the application of both NPAAs upregulated the three antioxidant-related genes PvCAT1, PvCuZnSOD1, and PvGR. Collectively, the fungistatic activity of NPAAs, coupled with their ability to alleviate oxidative stress, enhance antioxidant defenses, and stimulate plant growth, establishes them as promising eco-friendly alternatives for white mold disease management for sustainable bean production.

RNAseq-based transcriptome analysis of lettuce infected by the necrotrophic fungus Sclerotinia Sclerotiorum

RNAseq-based transcriptome analysis of lettuce infected by the necrotrophic fungus Sclerotinia Sclerotiorum

SsAGM1-Mediated Uridine Diphosphate-N-Acetylglucosamine Synthesis Is Essential for Development, Stress Response, and Pathogenicity of Sclerotinia sclerotiorum.

The necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen. S. sclerotiorum can cause Sclerotinia stem rot in more than 600 species of plants, which results in serious economic losses every year. Chitin is one of the most important polysaccharides in fungal cell walls. Chitin and β-Glucan form a scaffold that wraps around the cell and determines the vegetative growth and pathogenicity of pathogens. UDP-GlcNAc is a direct precursor of chitin synthesis. During the synthesis of UDP-GlcNAc, the conversion of GlcNAc-6P to GlcNAc-1P that is catalyzed by AGM1 (N-acetylglucosamine-phosphate mutase) is a key step. However, the significance and role of AGM1 in phytopathogenic fungus are unclear. We identified a cytoplasm-localized SsAGM1 in S. sclerotiorum, which is homologous to AGM1 of Saccharomyces cerevisiae. We utilized RNA interference (RNAi) and overexpression to characterize the function of SsAGM1 in S. sclerotiorum. After reducing the expression of SsAGM1, the contents of chitin and UDP-GlcNAc decreased significantly. Concomitantly, the gene-silenced transformants of SsAGM1 slowed vegetative growth and, importantly, lost the ability to produce sclerotia and infection cushion; it also lost virulence, even on wounded leaves. In addition, SsAGM1 was also involved in the response to osmotic stress and inhibitors of cell wall synthesis. Our results revealed the function of SsAGM1 in the growth, development, stress response, and pathogenicity in S. sclerotiorum.

Sclerotinia sclerotiorum Response to Long Exposure to Glucosinolate Hydrolysis Products by Transcriptomic Approach.

ABSTRACTWhite mold disease, caused by the necrotrophic fungus Sclerotinia sclerotiorum, affects Brassica crops. Brassica crops produce a broad array of compounds, such as glucosinolates, which contribute to the defense against pathogens. From their hydrolysis, several products arise that have antimicrobial activity (GHPs) whose toxicity is structure dependent. S. sclerotiorum may overcome the toxic effect of moderate GHP concentrations after prolonged exposure to their action. Our objective was to identify the molecular mechanism underlying S. sclerotiorum response to long exposure to two chemically diverse GHPs: aliphatic GHP allyl-isothiocyanate (AITC) and indole GHP indol-3-carbinol (I3C). We found that the transcriptomic response is dependent on the type of GHP and on their initial target, involving cell membranes in the case of AITC or DNA in the case of I3C. Response mechanisms include the reorganization of chromatin, mediated by histone chaperones hip4 and cia1, ribosome synthesis controlled by the kinase-phosphatase pair aps1-ppn1, catabolism of proteins, ergosterol synthesis, and induction of detoxification systems. These mechanisms probably help S. sclerotiorum to grow and survive in an environment where GHPs are constantly produced by Brassica plants upon glucosinolate breakdown.IMPORTANCE Brassica species, including important vegetable crops, such as cabbage, cauliflower, or broccoli, or oil crops, such as rapeseed, produce specific chemical compounds useful to protect them against pests and pathogens. One of the most destructive Brassica diseases in temperate areas around the world is Sclerotinia stem rot, caused by the fungus Sclerotinia sclerotiorum. This is a generalist pathogen that causes disease over more than 400 plant species, being a serious threat to economically important crops worldwide, including potato, bean, soybean, and sunflower, among many others. Understanding the mechanisms utilized by pathogens to overcome specific plant defensive compounds can be useful to increase plant resistance. Our study demonstrated that Sclerotinia shows different adaptation mechanisms, including detoxification systems, to grow and survive when plant protective compounds are present.

Exploring sunflower responses to Sclerotinia head rot at early stages of infection using RNA-seq analysis

Sclerotinia head rot (SHR), caused by the necrotrophic fungus Sclerotinia sclerotiorum, is one of the most devastating sunflower crop diseases. Despite its worldwide occurrence, the genetic determinants of plant resistance are still largely unknown. Here, we investigated the Sclerotinia-sunflower pathosystem by analysing temporal changes in gene expression in one susceptible and two tolerant inbred lines (IL) inoculated with the pathogen under field conditions. Differential expression analysis showed little overlapping among ILs, suggesting genotype-specific control of cell defense responses possibly related to differences in disease resistance strategies. Functional enrichment assessments yielded a similar pattern. However, all three ILs altered the expression of genes involved in the cellular redox state and cell wall remodeling, in agreement with current knowledge about the initiation of plant immune responses. Remarkably, the over-representation of long non-coding RNAs (lncRNA) was another common feature among ILs. Our findings highlight the diversity of transcriptional responses to SHR within sunflower breeding lines and provide evidence of lncRNAs playing a significant role at early stages of defense.

Rapid identification of an Arabidopsis NLR gene as a candidate conferring susceptibility to Sclerotinia sclerotiorum using time-resolved automated phenotyping.

SUMMARYThe broad host range necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen of many oil and vegetable crops. Plant genes conferring complete resistance against S. sclerotiorum have not been reported. Instead, plant populations challenged by S. sclerotiorum exhibit a continuum of partial resistance designated as quantitative disease resistance (QDR). Because of their complex interplay and their small phenotypic effect, the functional characterization of QDR genes remains limited. How broad host range necrotrophic fungi manipulate plant programmed cell death is for instance largely unknown. Here, we designed a time‐resolved automated disease phenotyping pipeline enabling high‐throughput disease lesion measurement with high resolution, low footprint at low cost. We could accurately recover contrasted disease responses in several pathosystems using this system. We used our phenotyping pipeline to assess the kinetics of disease symptoms caused by seven S. sclerotiorum isolates on six A. thaliana natural accessions with unprecedented resolution. Large effect polymorphisms common to the most resistant A. thaliana accessions identified highly divergent alleles of the nucleotide‐binding site leucine‐rich repeat gene LAZ5 in the resistant accessions Rubezhnoe and Lip‐0. We show that impaired LAZ5 expression in laz5.1 mutant lines and in A. thaliana Rub natural accession correlate with enhanced QDR to S. sclerotiorum. These findings illustrate the value of time‐resolved image‐based phenotyping for unravelling the genetic bases of complex traits such as QDR. Our results suggest that S. sclerotiorum manipulates plant sphingolipid pathways guarded by LAZ5 to trigger programmed cell death and cause disease.

Plant Growth Promotion and Biocontrol Potential of Fungal Endophytes in the Inflorescence of Aloe vera L.

Sclerotinia stem rot caused by necrotrophic plant pathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary is a highly pervasive disease affecting a wide range of agricultural and horticultural crops. In this study, fungal endophytes recovered from inflorescence tissues of Aloe vera L. were examined for their antagonist activity against S. sclerotiorum and the presence of plant growth-promoting traits. Fungal endophytes were characterized by microscopic and molecular (ITS rDNA sequencing) methods. Chaetomium globosum was the predominant endophytic species followed by Trichoderma harzanium in this study. Trichoderma harzanium tested positive for the presence of plant growth-promoting traits, i.e. siderophore, indole acetic acid, hydrogen cyanide production and phosphate solubilization. Chaetomium globosum strain 2 and T. harzanium survived stressful conditions (high temperature, cold stress and UV-B exposure). In antagonism assay, T. harzanium exhibited the highest growth inhibition per cent of 81.01 ± 0.63 against S. sclerotiorum and also restricted sclerotia formation. The hexane extract of T. harzanium (TH hex) obtained from solid fermentation depicted best antifungal activity against phytopathogen having an IC50 value of 3.987 mg/ml which is moderately comparable with positive control carbendazim (50%, WP). The GC–MS analysis of TH hex detected abundant presence (39.4%) of hexadecanoic acid, 2, 3-bis [(trimethylsilyl) oxy] propyl ester and other unsaturated fatty acids (palmitic and linoleic acid) having antifungal activity. The study demonstrates antifungal activity and plant growth-promoting potential of fungal endophytes residing in the inflorescence of A. vera.

Role of Major Glucosinolates in the Defense of Kale Against Sclerotinia sclerotiorum and Xanthomonas campestris pv. campestris.

Glucosinolates (GSLs) are secondary metabolites present in Brassicaceae species implicated in their defense against plant pathogens. When a pathogen causes tissue damage, the enzyme myrosinase hydrolyzes GSLs into diverse products that exhibit antimicrobial activity against a wide range of bacteria and fungi in vitro. It was demonstrated that modulation of GSL content in vivo affects plant resistance to infection by pathogens in Arabidopsis. However, the roles of specific metabolites and how they interact with pathogens are poorly understood in Brassica crops. We previously developed a set of populations of Brassica oleracea var. acephala L. (kale) differing in content of three GSLs: the aliphatics sinigrin (2-propenyl [SIN]) and glucoiberin (3-methylsulphinylpropyl [GIB]) and the indolic glucobrassicin (3-indolylmethyl [GBS]). These populations can be used to study the effects of major GSLs in kale, with the advantage that genotypes within each selection have the same genetic background. This research aimed to explore the role of SIN, GIB, and GBS in the defense of kale against the necrotrophic fungus Sclerotinia sclerotiorum and the bacterium Xanthomonas campestris pv. campestris. Results showed that increasing the amount of a particular GSL did not always result in disease resistance. The effects of GSLs were apparently dependent on the pathogen and the type of GSL. Thus, the aliphatic SIN was inhibitory to infection by S. sclerotiorum and the indolic GBS was inhibitory to infection by X. campestris pv. campestris. Other factors, including the quantity and proportion of other metabolites modified during the pathogen infection process, could also modulate the degree of inhibition to the pathogen.

The biocontrol agent Pseudomonas chlororaphis PA23 primes Brassica napus defenses through distinct gene networks

BackgroundThe biological control agent Pseudomonas chlororaphis PA23 is capable of protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. While we have elucidated bacterial genes and gene products responsible biocontrol, little is known about how the host plant responds to bacterial priming on the leaf surface, including global changes in gene activity in the presence and absence of S. sclerotiorum.ResultsApplication of PA23 to the aerial surfaces of canola plants reduced the number of S. sclerotiorum lesion-forming petals by 91.1%. RNA sequencing of the host pathogen interface showed that pretreatment with PA23 reduced the number of genes upregulated in response to S. sclerotiorum by 16-fold. By itself, PA23 activated unique defense networks indicative of defense priming. Genes encoding MAMP-triggered immunity receptors detecting flagellin and peptidoglycan were downregulated in PA23 only-treated plants, consistent with post-stimulus desensitization. Downstream, we observed reactive oxygen species (ROS) production involving low levels of H2O2 and overexpression of genes associated with glycerol-3-phosphate (G3P)-mediated systemic acquired resistance (SAR). Leaf chloroplasts exhibited increased thylakoid membrane structures and chlorophyll content, while lipid metabolic processes were upregulated.ConclusionIn addition to directly antagonizing S. sclerotiorum, PA23 primes the plant defense response through induction of unique local and systemic defense networks. This study provides novel insight into the effects of biocontrol agents applied to the plant phyllosphere. Understanding these interactions will aid in the development of biocontrol systems as an alternative to chemical pesticides for protection of important crop systems.

Genome-wide Association Study Identifies New Loci for Resistance to Sclerotinia Stem Rot in Brassica napus.

Sclerotinia stem rot (SSR) caused by the necrotrophic fungus Sclerotinia sclerotiorum is a major disease in rapeseed (Brassica napus) worldwide. Breeding for SSR resistance in B. napus, as in other crops, relies only on germplasms with quantitative resistance genes. A better understanding of the genetic basis for SSR resistance in B. napus thus holds promise for the genetic improvement of disease resistance. In the present study, a genome-wide association study (GWAS) for SSR resistance in B. napus were performed using an association panel of 448 accessions genotyped with the Brassica 60K Infinium® single-nucleotide polymorphism (SNP) array. A total of 26 SNPs corresponding to three loci, DSRC4, DSRC6, and DSRC8 were associated with SSR resistance. Haplotype analysis showed that the three favorable alleles for SSR resistance exhibited cumulative effects. After aligning SSR resistance quantitative trait loci (QTL) identified in the present and previous studies to the B. napus reference genome, one locus (DSRC6) was found to be located within the confidence interval of a QTL identified in previous QTL mapping studies and another two loci (DSRC4 and DSRC8) were considered novel loci for SSR resistance. A total of 39 candidate genes were predicted for the three loci based on the GWAS combining with the differentially expressed genes identified in previous transcriptomics analyses.

Histopathology of Sclerotinia sclerotiorum infection and oxalic acid function in susceptible and resistant soybean

The success of the necrotrophic fungus Sclerotinia sclerotiorum is largely dependent on its major virulence factor, oxalic acid (OA). Virulence is lost in transgenic plants that express OA degrading enzymes, e.g. oxalate oxidase (OxO). The histopathology of S. sclerotiorum infection and OA accumulation was examined in a transgenic soybean line over‐expressing OxO (OxO‐OE) and its isogenic parent (WT). In situ flower inoculation showed that the OxO‐OE plants were highly resistant to the pathogen while the WT parents were susceptible. This difference in resistance was not apparent in the floral tissues, as aggressive hyphal activity was similar on both hosts, showing that high OxO activity and low OA accumulation in OxO‐OE was not a deterrent. However, the process of fungal infection on excised leaf tissue differed on the two hosts. Primary lesions developed and showed similar severe ultrastructural damage on both hosts but rapid lesion expansion (colonization) proceeded only on the WT, concomitant with OA accumulation. Oxalic acid rose in OxO‐OE 1 day post‐inoculation and did not change over the following 3 days, showing that colonization can be blocked by maintaining low levels of OA. However, OxO degradation of OA did not deter initial host penetration and primary lesion formation. This shows that OA, the major virulence factor of S. sclerotiorum, is critical for host colonization but may not be required during primary lesion formation, suggesting that other factors are contributing to the establishment of the primary lesion.

The Microbial Opsin Homolog Sop1 is involved in Sclerotinia sclerotiorum Development and Environmental Stress Response.

Microbial opsins play a crucial role in responses to various environmental signals. Here, we report that the microbial opsin homolog gene sop1 from the necrotrophic phytopathogenic fungus Sclerotinia sclerotiorum was dramatically up-regulated during infection and sclerotial development compared with the vegetative growth stage. Further, study showed that sop1 was essential for growth, sclerotial development and full virulence of S. sclerotiorum. Sop1-silenced transformants were more sensitive to high salt stress, fungicides and high osmotic stress. However, they were more tolerant to oxidative stress compared with the wild-type strain, suggesting that sop1 is involved in different stress responses and fungicide resistance, which plays a role in the environmental adaptability of S. sclerotiorum. Furthermore, a Delta blast search showed that microbial opsins are absent from the genomes of animals and most higher plants, indicating that sop1 is a potential drug target for disease control of S. sclerotiorum.

Evaluation of Annual Bedding Plants for Resistance to White Mold

White mold, caused by the necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary, causes stem rot, crown rot, wilt, and death of many common herbaceous ornamental plants. Relatively little research has been done on management of white mold and resistance to this disease in ornamental crops. Plant varieties from four genera of widely available annual bedding plants with no reported history of white mold susceptibility were evaluated for potential resistance to S. sclerotiorum. All plants were inoculated with three isolates of S. sclerotiorum and evaluated for disease severity in a controlled environment and under field conditions. Portulaca grandiflora Hook. ‘Sundial Scarlet’, P. grandiflora ‘Sundial White’, Pentas lanceolata Forssk. ‘Graffiti Pink’, and Scaevola aemula R. Br. ‘Whirlwind White’ were highly susceptible to white mold in the controlled environment but had significantly lower disease incidence and severity than Zinnia elegans ×angustifolia ‘Profusion White’, the susceptible control, under field conditions. Impatiens hawkeri W. Bull. ‘Sonic Red’, I. hawkeri ‘Sonic Amethyst’, I. hawkeri ‘Sonic White’, I. walleriana Hook. f. ‘Blitz 3000 Red’, I. walleriana ‘Blitz 3000 White’, and I. walleriana ‘Blitz 3000 Rose’ displayed abscission of diseased plant tissue as an unusual resistance response. Plants from all four genera that were evaluated became infected with S. sclerotiorum to a lesser extent than susceptible controls under field conditions and could be used as part of an integrated disease management program for white mold in ornamental plantings.

Members of the germin-like protein family in Brassica napus are candidates for the initiation of an oxidative burst that impedes pathogenesis of Sclerotinia sclerotiorum

Germin-like proteins (GLPs) are defined by their sequence homology to germins from barley and are present ubiquitously in plants. Analyses of corresponding genes have revealed diverse functions of GLPs in plant development and biotic and abiotic stresses. This study describes the identification of a family of 14 germin-like genes from Brassica napus (BnGLP) designated BnGLP1–BnGLP14 and investigated potential functions of BnGLPs in plant defense against the necrotrophic fungus Sclerotinia sclerotiorum. Sequence alignment and phylogenetic analyses classify the 14 BnGLPs into four groups, which were clearly distinguished from known germin oxalic acid oxidases. Transcriptional responses of the BnGLP genes to S. sclerotiorum infection was determined by comparing cultivars of susceptible B. napus ‘Falcon’ and partially resistant B. napus ‘Zhongshuang 9’. Of the 14 BnGLP genes tested, BnGLP3 was transcriptionally upregulated in both B. napus cultivars at 6h after S. sclerotiorum infection, while upregulation of BnGLP12 was restricted to resistant B. napus ‘Zhongshuang 9’. Biochemical analysis of five representative BnGLP members identified a H2O2-generating superoxide dismutase activity only for higher molecular weight complexes of BnGLP3 and BnGLP12. By analogy, H2O2 formation at infected leaf sites increased after 6h, with even higher H2O2 production in B. napus ‘Zhongshuang 9’ compared with B. napus ‘Falcon’. Conversely, exogenous application of H2O2 significantly reduced the susceptibility of B. napus ‘Falcon’. These data suggest that early induction of BnGLP3 and BnGLP12 participates in an oxidative burst that may play a pivotal role in defence of B. napus against S. sclerotiorum.

Comparative QTL Map for White Mold Resistance in Common Bean, and Characterization of Partial Resistance in Dry Bean Lines VA19 and I9365‐3

White mold caused by the necrotrophic fungusSclerotinia sclerotiorum(Lib.) de Bary limits common bean (Phaseolus vulgarisL.) production in temperate climates. Disease resistance has been identified, but breeding is hampered by a paucity of resistance sources and complex inheritance, as numerous quantitative trait loci (QTL) conditioning partial resistance have been found. Our objectives were to characterize the partial white mold resistance found in breeding lines I9365‐31 and VA19 and to construct a comparative linkage map for all the white mold resistance QTL identified to date. Recombinant inbred line (RIL) populations ‘Benton’/VA19 (BV) and ‘Raven’/I9365‐31 (R31) consisting of 79 F6and 105 F5RILs, respectively, were evaluated for white mold (WM) reaction in multiple greenhouse and field tests. Two QTL were found in BV, WM2.2 expressed in the greenhouse (R2= 33%) and the field (13%) and WM8.3 expressed in field (11%) only, and seven were found in R31 (WM2.2, WM4.2, WM5.3, WM5.4, WM6.1, WM7.3, WM8.4), three expressed in greenhouse tests and four in the field, ranging in phenotypic variance from 5 to 52%. These QTL were compared with 26 previously identified QTL, resulting in a comparative linkage map of 35 QTL, which coalesced into 21 distinct regions across nine linkage groups. Four QTL found in R31 were novel. Sequence characterized amplified region markers associated with WM2.2, WM8.3, and WM7.3 QTL were generated. The comparative linkage map provides a framework for integrating and interpreting future QTL studies concerning white mold resistance in common bean.

Characterization of a canola C2 domain gene that interacts with PG, an effector of the necrotrophic fungus Sclerotinia sclerotiorum

Sspg1d, one of endopolygalacturonases, is an important fungal effector secreted by the necrotrophic fungus Sclerotinia sclerotiorum during early infection. Using sspg1d as bait, a small C2 domain protein (designated as IPG-1) was identified by yeast two-hybrid screening of a canola cDNA library. Deletion analysis confirmed that the C-terminus of IPG-1 is responsible for its interaction with sspg1d in the yeast two-hybrid assay. The sspg1d/IPG-1 interaction was further confirmed in plant cells by a biomolecular fluorescence complementation (BiFC) assay. A transient expression assay showed that the IPG-1–GFP fusion protein was targeted to the plasma membrane and nucleus in onion epidermal cells. Following treatment with a Ca2+ ionophore, it was distributed throughout the cytosol. Real-time PCR assay demonstrated that IPG-1 was highly induced by Sclerotinia sclerotiorum in canola leaves and stems. Southern blot analysis indicated the presence of about five homologues of IPG-1 in the canola genome. Two additional members of the IPG-1gene family were isolated by RT-PCR. Their sequence similarity with IPG-1 is as high as 95%. However, they did not interact with sspg1d in the yeast two-hybrid assay. Possible roles of IPG-1 and its association with sspg1d in the defence signalling pathway were discussed.

Apoplastic Polyamine Oxidation Plays Different Roles in Local Responses of Tobacco to Infection by the Necrotrophic FungusSclerotinia sclerotiorumand the Biotrophic BacteriumPseudomonas viridiflava

The role of polyamine (PA) metabolism in tobacco (Nicotiana tabacum) defense against pathogens with contrasting pathogenic strategies was evaluated. Infection by the necrotrophic fungus Sclerotinia sclerotiorum resulted in increased arginine decarboxylase expression and activity in host tissues, as well as putrescine and spermine accumulation in leaf apoplast. Enhancement of leaf PA levels, either by using transgenic plants or infiltration with exogenous PAs, led to increased necrosis due to infection by S. sclerotiorum. Specific inhibition of diamine and PA oxidases attenuated the PA-induced enhancement of leaf necrosis during fungal infection. When tobacco responses to infection by the biotrophic bacterium Pseudomonas viridiflava were investigated, an increase of apoplastic spermine levels was detected. Enhancement of host PA levels by the above-described experimental approaches strongly decreased in planta bacterial growth, an effect that was blocked by a PA oxidase inhibitor. It can be concluded that accumulation and further oxidation of free PAs in the leaf apoplast of tobacco plants occurs in a similar, although not identical way during tobacco defense against infection by microorganisms with contrasting pathogenesis strategies. This response affects the pathogen's ability to colonize host tissues and results are detrimental for plant defense against necrotrophic pathogens that feed on necrotic tissue; on the contrary, this response plays a beneficial role in defense against biotrophic pathogens that depend on living tissue for successful host colonization. Thus, apoplastic PAs play important roles in plant-pathogen interactions, and modulation of host PA levels, particularly in the leaf apoplast, may lead to significant changes in host susceptibility to different kinds of pathogens.

Reactive oxygen intermediates and oxalic acid in the pathogenesis of the necrotrophic fungus Sclerotinia sclerotiorum

An effective colonization of the host plant tissue by the necrotrophic fungus Sclerotinia sclerotiorum requires the secretion of the non-host specific toxin oxalic acid (OA), which is known to suppress the generation of reactive oxygen intermediates (ROI). A full-length cDNA coding for an oxalate decarboxylase (TOXDC), which converts OA into CO2 and formate, was isolated from the basidiomycete Trametes versicolor. It was overexpressed in tobacco plants to study the role of ROI and OA in the interaction between tobacco and S. sclerotiorum. The transgenic plants contained less OA and showed a delayed colonization of S. sclerotiorum; furthermore a strong ROI accumulation and nearly no catalase activity compared to the wild type (WT) plants could be detected. In addition, inoculation experiments with transgenic catalase-deficient plants (CAT1AS) and in vitro studies showed that S. sclerotiorum copes with strong ROI stress. Our results indicate that OA supports the infection process caused by S. sclerotiorum and the fungus itself is able to tolerate high ROI concentrations.