Two<Emphasis Type="Italic"> Brassica napus</Emphasis> polygalacturonase inhibitory protein genes are expressed at different levels in response to biotic and abiotic stresses

Plants encode a distinct set of polygalacturonase inhibitory proteins (PGIPs) that function to inhibit polygalacturonase enzymes produced by soft-rot fungal pathogens. We characterized two PGIP-encoding genes ( Bnpgip1 and Bnpgip2) from Brassica napus DH12075 (a double-haploid line derived from a cross between 'Crésor' and 'Westar'). The two proteins exhibit 67.4% identity at the amino acid level and contain 10 imperfect leucine-rich repeats. The pgip genes are present as a small multigene family in B. napus with at least four members. Bnpgip1 and Bnpgip2 are constitutively expressed in roots, stems, flower buds and open flowers. In mature leaf tissue, different levels of induction were observed in response to biotic and abiotic stresses. Bnpgip1 expression was highly responsive to flea beetle feeding and mechanical wounding, weakly responsive to Sclerotinia sclerotiorum infection and exposure to cold but not to dehydration. Conversely, Bnpgip2 expression was strongly induced by S. sclerotiorum infection and to a lesser degree by wounding but not by flea beetle feeding. Application of jasmonic acid to leaves induced both Bnpgip1 and Bnpgip2 gene expression; however, salicylic acid did not activate either gene. Taken together, these results suggest that separate pathways regulate Bnpgip1 and Bnpgip2, and that their roles in plant development or resistance to biotic and abiotic stress differ.     

Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis

Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungal pathogen capable of infecting a wide range of plants. To identify genes involved in fungal development and pathogenesis we generated 2232 expressed sequence tags (ESTs) from two cDNA libraries constructed using either mycelia grown in pectin medium or tissues from infected Brassica napus stems. A total of 774 individual fungal genes were identified of which 39 were represented only among the infected plant EST collection. Annotation of 534 unigenes was possible following the categories applied to Saccharomyces cerevisiae and the Universal Gene Ontology scheme. cDNAs were identified that encoded potential pathogenicity factors including four endopolygalacturonases, two exopolygalacturonases, and several metabolite transporters. The potential role of these genes, as well as those encoding signal transduction factors, in the infection process is discussed.     

Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction

Pseudomonas chlororaphis PA23, Pseudomonas spp. strain DF41, and Bacillus amyloliquefaciens BS6 consistently inhibit infection of canola petals by Sclerotinia sclerotiorum in both greenhouse and field experiments. Bacillus thuringiensis BS8, Bacillus cereus L, and Bacillus mycoides S have shown significant inhibition against S. sclerotiorum on plate assays. The presence of antibiotic biosynthetic or self-resistance genes in these strains was investigated with polymerase chain reaction and, in one case, Southern blotting. Thirty primers were used to amplify (i) antibiotic biosythetic genes encoding phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin, and (ii) the zwittermicin A self-resistance gene. Our findings revealed that the fungal antagonist P. chlororaphis PA23 contains biosynthetic genes for phenazine-1-carboxylic acid and pyrrolnitrin. Moreover, production of these compounds was confirmed by high performance liquid chromatography. Pseudomonas spp. DF41 and B. amyloliquefaciens BS6 do not appear to harbour genes for any of the antibiotics tested. Bacillus thuringiensis BS8, B. cereus L, and B. mycoides S contain the zwittermicin A self-resistance gene. This is the first report of zmaR in B. mycoides.     

Physiological and Molecular Features of the Pathosystem Arabidopsis thaliana L.-Sclerotinia sclerotiorum Libert

The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum and the molecular basis of plant responses to the pathogen are poorly understood. In the present investigation, the process of S. sclerotiorum infection in Arabidopsis thaliana L., a plant that is highly susceptible to this fungus, was analysed. In addition, the defense activation in the host was investigated. A convenient inoculation method using millet grain was developed for S. sclerotiorum in Arabidopsis. The fungus rapidly infected the plants, probably through ball- or cushion-like infection structures. Visible symptoms developed within 24 h and plants were killed 72 h after inoculation. Cellulase, the main enzyme that caused host tissues to rot, was secreted by S. sclerotiorum in a pH-dependent manner. Oxalic acid, another pathogenic factor secreted by the fungus, induced necrotic lesions on the leaves, infection with S. sclerotiorum strongly induced the production of the pathogenesis-related （PR） proteins β-1,3-glucanase and chitinase in Arabidopsis. Furthermore, the PR gene PDF. 1 was induced, but not PR1, indicating that the pathogen activated basal defense of jasmonic acid/ethylene dependence, which is consistent with its necrotrophic characteristics. This pathosystem for Arabidopsis-S. sclerotiorum could provide an approach for the analysis of the interactions between S. sclerotiorum and other crops, thereby facilitating genetic manipulation techniques for controlling this pathogen.     

<Emphasis Type="Italic">Brassica napus</Emphasis> possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> infection,wounding and defense hormone treatment

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form beta-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.     

Identification of prior candidate genes for Sclerotinia local resistance in Brassica napus using Arabidopsis cDNA microarray and Brassica-Arabidopsis comparative mapping

Arabidopsis was the leading model of dicot plant. An accomplished platform had been established for functional genomics studies. The platform had largely facilitated molecular biology research of Arabidopsis itself as well as research on its phylogenetic related plants[1,2]. Brassica napus, as an important cooking oil crop, had a close phylogenetic relationship with Arabidopsis[3―5]. In order to take advantage of avail-able Arabidopsis genetic and molecular tools, Girke et al. had explored …     

Type 2A phosphoprotein phosphatase is required for asexual development and pathogenesis of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Plant infection depends on the formation of melanin-rich infection cushions, and secretion of hydrolytic enzymes and oxalic acid. Type 2A Ser/Thr phosphatases (PP2As) are involved in the regulation of a variety of cellular process. In the presence of cantharidin, a PP2A-specific inhibitor, hyphal elongation and sclerotia numbers were impaired whereas sclerotial size increased. We partially inactivated PP2A by antisense expression of the gene (pph1) encoding the PP2A catalytic subunit. When antisense expression was induced, almost complete cessation of fungal growth was observed, indicative of a crucial role for PP2A in fungal growth. RNAi-based gene silencing was employed to alter the expression of the 55-kDa R2 (B regulatory subunit). Isolates in which rgb1 RNA levels were decreased were slow growing, but viable. Melanin biosynthesis, infection-cushion production, and pathogenesis were significantly impaired in the rgb1 mutants, yet theses mutants were pathogenic on wounded leaves. Reduced ERK (extracellular signal-regulated kinases)-like mitogen-activated protein kinase (MAPK) function conferred a reduction in NADPH oxidase and PP2A activity levels, suggesting a functional link between MAPK, reactive oxygen species, and PP2A activity in S. sclerotiorum.     

Indolyl-3-acetaldoxime dehydratase from the phytopathogenic fungus Sclerotinia sclerotiorum: purification, characterization, and substrate specificity

The purification and characterization of indolyl-3-acetaldoxime dehydratase produced by the plant fungal pathogen Sclerotinia sclerotiorum is described. The substrate specificity indicates that it is an indolyl-3-acetaldoxime dehydratase (IAD, EC 4.99.1.6), which catalyzes transformation of indolyl-3-acetaldoxime to indolyl-3-acetonitrile. The enzyme showed Michaelis-Menten kinetics and had an apparent molecular mass of 44 kDa. The amino acid sequence of IAD, determined using LC-ESI-MS/MS, identified it as the protein SS1G_01653 from S. sclerotiorum. IADSs was highly homologous (84% amino acid identity) to the hypothetical protein BC1G_14775 from Botryotinia fuckeliana B05.10. In addition, similarity to the phenylacetaldoxime dehydratases from Gibberella zeae (33% amino acid identity) and Bacillus sp. (20% amino acid identity) was noted. The specific activity of IADSs increased about 17-fold upon addition of Na(2)S(2)O(4) under anaerobic conditions, but in the absence of Na(2)S(2)O(4) no significant change was observed, whether aerobic or anaerobic conditions were used. As with other aldoxime dehydratases isolated from microbes, the role of IADSs in fungal plant pathogens is not clear, but given its substrate specificity, it appears unlikely that IADSs is a general xenobiotic detoxifying enzyme.     

Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum

Homologous recombination is required for gene-targeted procedures such as gene disruption and gene replacement. Ku80 is part of the non-homologous end-joining DNA repair mechanism in many organisms. We identified and disrupted the Ku80 homologue in Sclerotinia sclerotiorum and generated heterokaryon mutants enriched with Ku80 -deficient nuclei ( ssku80 ). Sclerotial formation and pathogenicity of ssku80 mutants were normal on tomato fruits. The frequencies of homologous recombination in these strains were much higher than those of the wild type when transformed with a cna1 (encoding calcineurin) replacement construct. We coupled the increase in homologous recombination with a direct BIM-LAB-mediated transformation procedure, which utilizes compressed air to assist the transforming DNA in penetrating fungal hyphae of S. sclerotiorum . We found this method to be efficient and reproducible, and it did not alter the fitness of the mutants. We also demonstrated the first case of direct transformation of sclerotia. Nourseothricin was introduced as a selectable marker in S. sclerotiorum . The tools and procedures described will improve our ability to study gene function in S. sclerotiorum and are most likely to be adaptable for use in other plant pathogens.     

Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection

Oxalic acid is a virulence factor of several phytopathogenic fungi, including Sclerotinia sclerotiorum (Lib.) de Bary, but the detailed mechanisms by which oxalic acid affects host cells and tissues are not understood. We tested the hypothesis that oxalate induces foliar wilting during fungal infection by manipulating guard cells. Unlike uninfected leaves, stomatal pores of Vicia faba leaves infected with S. sclerotiorum are open at night. This cellular response appears to be dependent on oxalic acid because stomatal pores are partially closed when leaves are infected with an oxalate-deficient mutant of S. sclerotiorum. In contrast to oxalate-deficient S. sclerotiorum, wild-type fungus causes an increase in stomatal conductance and transpiration as well as a decrease in plant biomass. Green fluorescent protein-tagged S. sclerotiorum emerges through open stomata from the uninfected abaxial leaf surface for secondary colonization. Exogenous application of oxalic acid to the detached abaxial epidermis of V. faba leaves induces stomatal opening. Guard cells treated with oxalic acid accumulate potassium and break down starch, both of which are known to contribute to stomatal opening. Oxalate interferes with abscisic acid (ABA)-induced stomatal closure. The Arabidopsis (Arabidopsis thaliana) L. Heynh. mutants abi1, abi3, abi4, and aba2 are more susceptible to oxalate-deficient S. sclerotiorum than wild-type plants, suggesting that Sclerotinia resistance is dependent on ABA. We conclude that oxalate acts via (1) accumulation of osmotically active molecules to induce stomatal opening and (2) inhibition of ABA-induced stomatal closure.     

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.     

杆状病毒p35基因诱导烟草产生广谱抗病机理分析

来源于昆虫病毒和动物的抗细胞凋亡基因能够诱导植物对生物或者非生物胁迫产生抗性.但其抗性机理有不同甚至相反的报道.本研究将来源于苜蓿银纹夜蛾核多角体病毒的p35基因转化烟草,T1代转化烟草Western blotting检测P35蛋白的表达,转化烟草接种烟草花叶病毒(Tobacco mosaic virus,TMV)抗病效果增强.进一步的抗病机理研究表明,转化和野生型烟草感染TMV后诱导过氧化氢积累无明显区别,野生型烟草感染24 h后出现DNA Laddering而转化烟草则没有;Western blotting结果显示PR-1蛋白表达没有显著差异.但接种另外一种病原真菌核盘茵(Sclerotiniasclerotiorum)后的RT-PCR分析结果表明,表达P35蛋白的烟草可增强感染核盘菌后PR-1基因的转录.而且表达时间提前.以上结果说明p35基因介导的广谱抗病反应的机理与接种的不同病原有关,对不同病原物的抗病机理存在差异,除抑制细胞凋亡外,还可能通过激活PR基因的表达提高对病原物的抗病能力.