Identification of Botrytis cinerea susceptibility loci in Arabidopsis thaliana

Botrytis cinerea is a major pathogen of fruit and vegetable crops causing both pre- and post-harvest grey mould. We have analysed 16 Arabidopsis thaliana ecotypes for natural variation in B. cinerea susceptibility. Susceptibility was associated with lower camalexin accumulation, and three ecotypes (Cape Verdi Islands (Cvi-0), Slavice (Sav-0) and Kindalville (Kin-0)) showed differential susceptibility to the two B. cinerea isolates used. Subsequently, to better understand the genetic control of grey mould disease, we assayed the Arabidopsis Landsberg erecta (Ler) x Columbia (Col-0) recombinant inbred population with the two isolates, and identified multiple small-to-medium-effect quantitative trait loci (QTL) governing susceptibility. Interestingly, the QTL for each isolate are distinct, suggesting that different mechanisms govern defence against these two isolates. Two QTL for each isolate exhibited epistatic interactions with specific allele combinations generating heightened B. cinerea susceptibility.     

The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases

Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhibitory activity has not yet been linked conclusively to the activity of the Vvpgip1 gene product. Here we use a transgenic over-expression approach to show that the PGIP encoded by the Vvpgip1 gene is active against PGs of B. cinerea and that over-expression of this gene in transgenic tobacco confers a reduced susceptibility to infection by this pathogen. A calculated reduction in disease susceptibility of 47–69% was observed for a homogeneous group of transgenic lines that was statistically clearly separated from untransformed control plants following infection with Botrytis over a 15-day-period. VvPGIP1 was subsequently purified from transgenic tobacco and used to study the specific inhibition profile of individual PGs from Botrytis and Aspergillus. The heterologously expressed and purified VvPGIP1 selectively inhibited PGs from both A. niger and B.␣cinerea, including BcPG1, a PG from B. cinerea that has previously been shown to be essential for virulence and symptom development. Altogether our data confirm the antifungal nature of the VvPGIP1, and the in vitro inhibition data suggest at least in part, that the VvPGIP1 contributed to the observed reduction in disease symptoms by inhibiting the macerating action of certain Botrytis PGs in planta. The ability to correlate inhibition profiles to individual PGs provides a more comprehensive analysis of PGIPs as antifungal genes with biotechnological potential, and adds to our understanding of the importance of PGIP:PG interactions during disease and symptom development in plants.Dirk A. Joubert and Ana R. Slaughter contributed equally to this work.     

Fungal elicitor protein PebC1 from Botrytis cinerea improves disease resistance in Arabidopsis thaliana

We previously identified a novel protein elicitor, PebC1, from Botrytis cinerea and described its enhancement of plant growth, drought tolerance and disease resistance in tomato. Here, we have investigated the defense-associated molecular responses in Arabidopsis thaliana after treatment with recombinant PebC1. PebC1 was expressed in Escherichia coli. Recombinant protein treatments improved plant resistance to Botrytis infection and maintained plant defenses for more than 21 days. The purified protein at 10 μg ml^?1 activated extracellular medium alkalization (pH) and reactive oxygen species and nitric oxide generation and also induced defense gene expression. Arabidopsis mutants that are insensitive to salicylic acid had increased resistance to Botrytis infection after PebC1 treatment but PebC1 did not affect the resistance of mutants with jasmonic acid and ethylene transduction pathways. The results suggest that PebC1 can function as an activator of plant disease resistance and can promote disease resistance to Botrytis in A. thaliana through the ethylene signal transduction pathway.     

拟南芥新型脂转移蛋白AtDHyPRP1的亚细胞定位及其对灰霉菌的抗性

通过遗传转化技术研究了拟南芥脂转移蛋白AtDHyPRP1在细胞中的定位及其对真菌病原体的抗性。采用PCR方法从拟南芥Ws生态型克隆了AtDHyPRP1基因,构建产生pRI101-AN-AtDHyPRP1植物双元表达载体和pCAMBIA1302-AtDHyPRP1-GFP融合表达载体,经农杆菌介导的叶盘和浸花法得到烟草和拟南芥转基因植株。AtDHyPRP1基因能够明显增加烟草对灰霉菌的抗性,转AtDHyPRP1烟草叶片的被侵染部位有大量H2O2积累,激光共聚焦显微观察发现AtDHyPRP1蛋白定位于细胞表面。说明AtDHyPRP1蛋白在合成后被分泌到细胞外执行特殊的功能,与植物抗病防御机制有关。     

Complex genetics control natural variation in Arabidopsis thaliana resistance to Botrytis cinerea

The genetic architecture of plant defense against microbial pathogens may be influenced by pathogen lifestyle. While plant interactions with biotrophic pathogens are frequently controlled by the action of large-effect resistance genes that follow classic Mendelian inheritance, our study suggests that plant defense against the necrotrophic pathogen Botrytis cinerea is primarily quantitative and genetically complex. Few studies of quantitative resistance to necrotrophic pathogens have used large plant mapping populations to dissect the genetic structure of resistance. Using a large structured mapping population of Arabidopsis thaliana, we identified quantitative trait loci influencing plant response to B. cinerea, measured as expansion of necrotic lesions on leaves and accumulation of the antimicrobial compound camalexin. Testing multiple B. cinerea isolates, we identified 23 separate QTL in this population, ranging in isolate-specificity from being identified with a single isolate to controlling resistance against all isolates tested. We identified a set of QTL controlling accumulation of camalexin in response to pathogen infection that largely colocalized with lesion QTL. The identified resistance QTL appear to function in epistatic networks involving three or more loci. Detection of multilocus connections suggests that natural variation in specific signaling or response networks may control A. thaliana-B. cinerea interaction in this population.     

Absence of the endo-beta-1,4-glucanases Cel1 and Cel2 reduces susceptibility to Botrytis cinerea in tomato

Cel1 and Cel2 are members of the tomato (Solanum lycopersicum Mill) endo-beta-1,4-glucanase (EGase) family that may play a role in fruit ripening and organ abscission. This work demonstrates that Cel1 protein is present in other vegetative tissues and accumulates during leaf development. We recently reported the downregulation of both the Cel1 mRNA and protein upon fungal infection, suggesting the involvement of EGases in plant-pathogen interactions. This hypothesis was confirmed by assessing the resistance to Botrytis cinerea infection of transgenic plants expressing both genes in an antisense orientation (Anti-Cel1, Anti-Cel2 and Anti-Cel1-Cel2). The Anti-Cel1-Cel2 plants showed enhanced resistance to this fungal necrotroph. Microscopical analysis of infected leaves revealed that tomato plants accumulated pathogen-inducible callose within the expanding lesion. Anti-Cel1-Cel2 plants presented a faster and enhanced callose accumulation against B. cinerea than wild-type plants. The inhibitor 2-deoxy-d-glucose, a callose synthesis inhibitor, showed a direct relationship between faster callose accumulation and enhanced resistance to B. cinerea. EGase activity appears to negatively modulate callose deposition. The absence of both EGase genes was associated with changes in the expression of the pathogen-related genes PR1 and LoxD. Interestingly, Anti-Cel1-Cel2 plants were more susceptible to Pseudomonas syringae, displaying severe disease symptoms and enhanced bacterial growth relative to wild-type plants. Analysis of the involvement of Cel1 and Cel2 in the susceptibility to B. cinerea in fruits was done with the ripening-impaired mutants Never ripe (Nr) and Ripening inhibitor (rin). The data reported in this work support the idea that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens.     

Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea

Arabidopsis possesses two arginase-encoding genes, ARGAH1 and ARGAH2, catalysing the catabolism of arginine into ornithine and urea. Arginine and ornithine are both precursors for polyamine biosynthetic pathways. We observed an accumulation of ARGAH2 mRNA in Arabidopsis upon inoculation with the necrotrophic pathogen Botrytis cinerea. Transgenic lines displaying either overexpression of ARGAH2 or simultaneous silencing of both Arabidopsis arginase-encoding genes were created and their resistance to B. cinerea infection evaluated. Overexpression of arginase resulted in changes in amino acid accumulation, while polyamine levels remained largely unaffected. Silencing lines were affected in both amino acid and putrescine accumulation. Arabidopsis plants overexpressing the arginase gene were less susceptible to B. cinerea, whereas silencing lines remained as susceptible as the wild type. We discuss how arginase might interact with plant defence mechanisms. These results provide new insights into amino acid metabolic changes under stress.     

The role of phytohormones in basal resistance and Trichoderma-induced systemic resistance to Botrytis cinerea in Arabidopsis thaliana

Thirty-six phytohormone-affected mutants of Arabidopsis thaliana (L.) Heynh. and their parental ecotypes were tested for resistance/susceptibility to Botrytis cinerea Pers.; Fr. and ability to develop Trichoderma-mediated induced systemic resistance (ISR). Ecotype Colombia-0 (Col-0) was relatively resistant to B. cinerea, and Trichoderma harzianum Rifai T39 application at sites spatially separated (roots) from the B. cinerea inoculation (leaves) resulted in reduction of grey mold symptoms. Ecotypes Wassilewskija-4, Nossen-0 and Landsberg-0 had low levels of basal resistance to B. cinerea and were unable to express ISR. Mutants derived from ISR-non-inducible ecotypes displayed ISR-non-inducible phenotypes, whereas the ISR inducibility of mutants derived from the ISR-inducible genotype Col-0 varied according to the type of mutant. Thus, salicylic acid (SA)-impaired mutants derived from Col-0 were ISR-inducible, while ethylene/jasmonic acid (ethylene/JA)-impaired mutants of the same origin were ISR-non-inducible. SA-impaired mutants retained basal level of resistance to B. cinerea, while most ethylene/JA-impaired mutants were highly susceptible. Abscisic acid- and gibberellin-impaired mutants were highly susceptible to B. cinerea and showed ISR-non-inducible phenotypes irrespective of their lines of origin. Auxin-resistant mutants derived from Col-0 were ISR-inducible; mutant originating from Landsberg-0 and mutants which were resistant to both auxin and ethylene were ISR-non-inducible. Most of the arabidopsis genotypes which were unable to express Trichoderma-mediated ISR against B. cinerea exhibited enhanced susceptibility to this pathogen. T. harzianum treatments enhanced the growth of arabidopsis plants regardless of genotype or ISR inducibility.     

Arabidopsis ssi2-conferred susceptibility to Botrytis cinerea is dependent on EDS5 and PAD4

Loss of a stearoyl-ACP desaturase activity in the Arabidopsis thaliana ssi2 mutant confers susceptibility to the necrotroph, Botrytis cinerea. In contrast, the ssi2 mutant exhibits enhanced resistance to Pseudomonas syringae, Peronospora parasitica, and Cucumber mosaic virus. The altered basal resistance to these pathogens in the ssi2 mutant plant is accompanied by the constitutive accumulation of elevated salicylic acid (SA) level and expression of the pathogenesis-related 1 (PR1) gene, the inability of jasmonic acid (JA) to activate expression of the defensin gene, PDF1.2, and the spontaneous death of cells. Here, we show that presence of the eds5 and pad4 mutant alleles compromises the ssi2-conferred resistance to Pseudomonas syringae pv. maculicola. In contrast, resistance to B. cinerea was restored in the ssi2 eds5 and ssi2 pad4 double-mutant plants. However, resistance to B. cinerea was not accompanied by the restoration of JA responsiveness in the ssi2 eds5 and ssi2 pad4 plants. The ssi2 eds5 and ssi2 pad4 plants retain the ssi2-conferred spontaneous cell death phenotype, suggesting that cell death is not a major factor that predisposes the ssi2 mutant to infection by B. cinerea. Furthermore, the high SA content of the ssi2 pad4 plant, combined with our previous observation that the SA-deficient ssi2 nahG plant succumbs to infection by B. cinerea, suggests that elevated SA level does not have a causal role in the ssi2-conferred susceptibility to B. cinerea. Our results suggest that interaction between an SSI2-dependent factor or factors and an EDS5- and PAD4-dependent mechanism or mechanisms modulates defense to B. cinerea.     

Nitrogen availability and susceptibility of tomato leaves to Botrytis cinerea

The aim of this study was to investigate the effect of nitrogen availability on susceptibility of tomato leaves to the fungal pathogen Botrytis cinerea. Plants with varying nitrogen availability were grown by adding N daily in exponentially increasing amounts to a nutrient solution at different rates. Leaves of plants grown at low nitrogen availability had a high leaf C/N ratio (21 g g^-1) and were about 2.5 times more susceptible to primary lesion formation by B. cinerea compared to plant grown at high nitrogen availability, which had a low leaf C/N ratio (11 g g^-1). Leaf C/N ratio accounted for 95% of variation in susceptibility. This relationship between C/N ratio and susceptibility persisted when plants were grown with exponential P addition and optimal N supply, and was thus independent of plant growth rate or related factors. We could not explain the effect of nitrogen availability by variation in the most obvious N-based resistance compound α-tomatine because more susceptible leaves with a high C/N ratio contained more α-tomatine. These leaves also contained more soluble carbohydrates. The level of soluble carbohydrates correlated positively with susceptibility, independent of the growth method. We therefore suggest that the effect of N availability on susceptibility must be explained by variation in levels of soluble carbohydrates and speculate about the role of these carbohydrates in the infection process. This revised version was published online in June 2006 with corrections to the Cover Date.     

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.     

Host-induced gene silencing of BcTOR in Botrytis cinerea enhances plant resistance to grey mould

Botrytis cinerea is the causal agent of grey mould for more than 200 plant species, including economically important vegetables, fruits and crops, which leads to economic losses worldwide. Target of rapamycin (TOR) acts a master regulator to control cell growth and proliferation by integrating nutrient, energy and growth factors in eukaryotic species, but little is known about whether TOR can function as a practicable target in the control of plant fungal pathogens. Here, we characterize TOR signalling of B. cinerea in the regulation of growth and pathogenicity as well as its potential value in genetic engineering for crop protection by bioinformatics analysis, pharmacological assays, biochemistry and genetics approaches. The results show that conserved TOR signalling occurs, and a functional FK506-binding protein 12 kD (FKBP12) mediates the interaction between rapamycin and B. cinerea TOR (BcTOR). RNA sequencing (RNA-Seq) analysis revealed that BcTOR displayed conserved functions, particularly in controlling growth and metabolism. Furthermore, pathogenicity assay showed that BcTOR inhibition efficiently reduces the infection of B. cinerea in plant leaves of Arabidopsis and potato or tomato fruits. Additionally, transgenic plants expressing double-stranded RNA of BcTOR through the host-induced gene silencing method could produce abundant small RNAs targeting BcTOR, and significantly block the occurrence of grey mould in potato and tomato. Taken together, our results suggest that BcTOR is an efficient target for genetic engineering in control of grey mould, and also a potential and promising target applied in the biocontrol of plant fungal pathogens.