The BOS loci of Arabidopsis are required for resistance to Botrytis cinerea infection

Three Botrytis-susceptible mutants bos2, bos3, and bos4 which define independent and novel genetic loci required for Arabidopsis resistance to Botrytis cinerea were isolated. The bos2 mutant is susceptible to B. cinerea but retains wild-type levels of resistance to other pathogens tested, indicative of a defect in a response pathway more specific to B. cinerea. The bos3 and bos4 mutants also show increased susceptibility to Alternaria brassicicola, another necrotrophic pathogen, suggesting a broader role for these loci in resistance. bos4 shows the broadest range of effects on resistance, being more susceptible to avirulent strain of Pseudomonas syringae pv. tomato. Interestingly, bos3 is more resistant than wild-type plants to virulent strains of the biotrophic pathogen Peronospora parasitica and the bacterial pathogen P. syringae pv. tomato. The Pathogenesis Related gene 1 (PR-1), a molecular marker of the salicylic acid (SA)-dependent resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3 this gene was expressed at elevated levels, both constitutively and in response to pathogen challenge. In bos4 plants, PR-1 expression was reduced compared with wild type in response to B. cinerea and SA. In bos3, the mutant most susceptible to B. cinerea and with the highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but no change to the B. cinerea response. Expression of the plant defensin gene PDF1-2 was generally lower in bos mutants compared with wild-type plants, with a particularly strong reduction in bos3. Production of the phytoalexin camalexin is another well-characterized plant defense response. The bos2 and bos4 mutants accumulate reduced levels of camalexin whereas bos3 accumulates significantly higher levels of camalexin than wild-type plants in response to B. cinerea. The BOS2, BOS3, and BOS4 loci may affect camalexin levels and responsiveness to ethylene and jasmonate. The three new mutants appear to mediate disease responses through mechanisms independent of the previously described BOS1 gene. Based on the differences in the phenotypes of the bos mutants, it appears that they affect different points in defense response pathways.     

Autophagy is required for tolerance of drought and salt stress in plants

Autophagy is a protein degradation process in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon the induction of autophagy, a double membrane autophagosome forms around cytoplasmic components and delivers them to the vacuole or lysosome for degradation. In plants, autophagy has been shown previously to be induced during abiotic stresses including nutrient starvation and oxidative stress. In this paper, we demonstrate the induction of autophagy in high salt and osmotic stress conditions, concomitant with the upregulation of expression of an Arabidopsis thaliana autophagy-related gene AtATG18a. Autophagy-defective RNAi-AtATG18a plants are more sensitive to salt and drought conditions than wild-type plants, demonstrating a role for autophagy in the response to these stresses. NADPH oxidase inhibitors block autophagy induction upon nutrient starvation and salt stress, but not during osmotic stress, indicating that autophagy can be activated by NADPH oxidase-dependent or -independent pathways. Together our results indicate that diverse environmental stresses can induce autophagy and that autophagy is regulated by distinct signaling pathways in different conditions.     

Mutational tolerance to carbendazim in Botrytis cinerea

No spontaneous mutation for tolerance to the fungicide carbendazim was detected in C. 10⁸ conidia from each of eight carbendazim-sensitive field isolates of Botrytis cinerea. Conidia of B. cinerea were highly insensitive to u.v.-irradiation, although after severe irradiation treatments mutant strains showing the same levels of tolerance as two groups of carbendazim-tolerant field isolates were selected at frequencies of between 10^-9 and 10^-6 of survivors. Mutants with low levels of tolerance (ED₅₀ > 10 μg ml^-1 carbendazim; ‘partially-tolerant’) were selected from irradiated conidia obtained from sensitive field isolates and a further series of mutants capable of growth on 10 000 μg ml^-1 carbendazim (‘fully-tolerant’) were selected from irradiated conidia from either partially-tolerant mutants or from partially-tolerant field isolates. Both mutation steps were confirmed in similar experiments in which tolerance to an unrelated fungicide, 2, 6-dichloro-4-nitroaniline (DCNA), was incorporated as a genetic marker in the parent strains.     

拟南芥灰霉病抗性相关转录因子

病原菌的侵染激发植物大量防御响应基因的表达, 其中转录因子在协调庞大的抗病防御网络中发挥重要作用。灰葡萄孢菌(Botrytis cinerea)是最具破坏力的死体营养型病原真菌之一, 在农业生产上造成严重的经济损失。文章综述了ERF(Ethylene response factors)、WRKY、MYB等家族中参与灰霉病防御反应的转录因子的功能研究进展。转录因子通过复杂的mRNA或蛋白水平的互作方式构成了精细的调控网络, 以激活下游防卫基因的表达, 从而诱导抗病反应。一部分转录因子是协调不同激素信号通路交叉响应的重要节点和调节器, 将植物抵御不同类型病原菌的分子机制联系起来。对这类转录因子的研究将为研究植物其他病原菌防御机制提供线索, 另外深入理解抗病机制将有助于研究者在作物改良和保护中更高效地利用抗病基因。     

The mitogen-activated protein kinase kinase kinase BcOs4 is required for vegetative differentiation and pathogenicity in Botrytis cinerea

The high-osmolarity glycerol signal pathway plays an important role in the response of fungi to various environmental stresses. In this study, we characterized a mitogen-activated protein kinase kinase kinase gene BcOS4 in Botrytis cinerea, which is homologous to Saccharomyces cerevisiae SSK2/SSK22. The BcOS4 deletion mutant was significantly impaired in vegetative growth and conidial formation. The mutant exhibited increased sensitivity to the osmotic, oxidative stresses and to the fungicides iprodione and fludioxonil. Western blot analysis showed that BcSak1, a putative downstream component of BcOs4, was not phosphorylated in the mutant. In addition, the BcOS4 mutant was unable to infect leaves of rapeseed and cucumber, and grape fruits, although it can cause disease on apple fruits. All the defects were restored by genetic complementation of the BcOS4 deletion mutant with the wild-type BcOS4 gene. The data of this study indicate that BcOS4 is involved in vegetative differentiation, virulence, adaption to hyperosmotic and oxidative stresses, and to fungicides in B. cinerea.     

The pepper extracellular peroxidase CaPO2 is required for salt, drought and oxidative stress tolerance as well as resistance to fungal pathogens

In plants, biotic and abiotic stresses regulate the expression and activity of various peroxidase isoforms. Capsicum annuum EXTRACELLULAR PEROXIDASE 2 (CaPO2) was previously shown to play a role in local and systemic reactive oxygen species bursts and disease resistance during bacterial pathogen infection. Here, we report CaPO2 expression patterns and functions during conditions of biotic and abiotic stress. In pepper plants, CaPO2 expression was strongly induced by abscisic acid, but not by defense-related plant hormones such as salicylic acid, ethylene and jasmonic acid. CaPO2 was also strongly induced by abiotic and biotic stress treatments, including drought, cold, high salinity and infection by the hemibiotrophic fungal pathogen Colletotrichum coccodes. Loss-of-function of CaPO2 in virus-induced gene silenced pepper plants led to increased susceptibility to salt- and osmotic-induced stress. In contrast, CaPO2 overexpression in transgenic Arabidopsis thaliana plants conferred enhanced tolerance to high salt, drought, and oxidative stress, while also enhancing resistance to infection by the necrotrophic fungal pathogen Alternaria brassicicola. Taken together, these results provide evidence for the involvement of pepper extracellular peroxidase CaPO2 in plant defense responses to various abiotic stresses and plant fungal pathogens.     

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

A putative bifunctional xylosidase/arabinofuranisodase localizes to the apoplast and is important for immunity against the necrotrophic pathogen Botrytis cinerea.     

The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-beta-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A, coding for an endo-beta-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-beta-1-4-xylanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.     

The response regulator BcSkn7 is required for vegetative differentiation and adaptation to oxidative and osmotic stresses in Botrytis cinerea

The high‐osmolarity glycerol pathway plays an important role in the responses of fungi to various environmental stresses. Saccharomyces cerevisiae Skn7 is a response regulator in the high‐osmolarity glycerol pathway, which regulates the oxidative stress response, cell cycle and cell wall biosynthesis. In this study, we characterized an Skn7 orthologue BcSkn7 in Botrytis cinerea. BcSKN7 can partly restore the growth defects of S. cerevisiae SKN7 mutant and vice versa. The BcSKN7 mutant (ΔBcSkn7‐1) revealed increased sensitivity to ionic osmotic and oxidative stresses and to ergosterol biosynthesis inhibitors. In addition, ΔBcSkn7‐1 was also impaired dramatically in conidiation and sclerotial formation. Western blot analysis showed that BcSkn7 positively regulated the phosphorylation of BcSak1 (the orthologue of S. cerevisiae Hog1) under osmotic stress, indicating that BcSkn7 is associated with the high‐osmolarity glycerol pathway in B. cinerea. In contrast with BcSak1, BcSkn7 is not involved in the regulation of B. cinerea virulence. All of the phenotypic defects of ΔBcSkn7‐1 are restored by genetic complementation of the mutant with the wild‐type BcSKN7. The results of this study indicate that BcSkn7 plays an important role in the regulation of vegetative differentiation and in the response to various stresses in B. cinerea.     

Overexpression of OgPAE1 from wild rice confers fungal resistance against Botrytis cinerea

A full-length cDNA of the OgPAE1 gene encoding the alpha5 subunit of the 20S proteasome was isolated from wild rice (Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism.     

Factors affecting the resistance of cold-stored carrots to Botrytis cinerea

The secondary phloem parenchyma of cold-stored turgid roots of carrot (Daucus carota) is capable of localizing mycelial infection by Botrytis cinerea, producing a dark resistant lesion. The percentage of roots exhibiting this reaction declined with increasing time in cold-store: when freshly harvested and wound-inoculated in October 1974, 99%of roots resisted invasion, whereas only 5% of those stored until March 1975 did so. The surface dimensions of resistant lesions did not increase between 33 and 55 days after inoculation. However, the surface dimensions and mean weights of lesions (arising from inoculations performed at different times over the course of the storage season) were both larger with increasing time in storage of roots prior to inoculation. The ability of the root tissue to localize infection was reduced if the roots lost 5–10% or more of their fresh weight before inoculation, resulting progressively in susceptibility. Compared with roots wound-inoculated using mycelial disks, there was an overall reduction in infection when carrots were wound-inoculated using conidia or when conidia or mycelial disks were inoculated onto the apparently undamaged surface of roots.     

An autonomously replicating plasmid transforms Botrytis cinerea to phleomycin resistance

Abstract A transformation system has been developed for the pathogen fungus Botrytis cinerea , based on the utilization of the wide host plasmid pUT737 that contains the Sh ble gene, conferring resistance to phleomycin. Transformed protoplasts were regenerated at 10–25 μg ml⁻¹ of phleomycin, at a frequency of 25–40 transformants per μg of DNA, and they were resistant up to 50 μg ml⁻¹. Southern hybridization using undigested and digested total DNA showed the presence of circular autonomously replicating plasmid pUT737 in the transformants. Reisolated plasmid from transformed fungus transformed E. coli and rescued plasmid was identified as pUT737. Transformants were grown for four generations under non-selective conditions and replicative plasmids were still detected. Plasmids present in all transformants at this stage had been modified from native pUT737 and showed the same size and configuration indicating that selection through stabilizing plasmid forms has happened.     

The phenylpropanoid pathway affects apple fruit resistance to Botrytis cinerea

Apple fruits are rich in phenolic compounds that may enhance resistance to grey mould disease caused by Botrytis cinerea. Using Malus domestica Borkh. cultivars Fuji and Qinguan, we analysed the contents of total phenols, total flavonoids, eight individual phenolic compounds, H₂O₂ and O₂^.? as well as the activities of key enzymes in the phenylpropanoid pathway in the flesh of control and B. cinerea‐inoculated fruits. Chlorogenic acid contents increased for a short period in the less susceptible cultivar Qinguan fruits, but decreased in the disease‐susceptible Fuji fruits. Additionally, ferulic acid production was induced in both cultivars in response to B. cinerea. Furthermore, the activities of phenylalanine ammonia lyase, cinnamate 4‐hydroxylase, 4‐coumarate:coenzyme A ligase and cinnamyl alcohol dehydrogenase were differentially induced between the two apple cultivars. Remarkably, the contents of H₂O₂ and O₂^.? as well as the activities of enzymes in phenolic metabolism tested in this study were always higher in Qinguan fruits than in Fuji fruits. Our data imply that phenylpropanoid metabolism is closely associated with apple fruit resistance to grey mould disease. These findings may be useful for characterizing the mechanism(s) underlying plant resistance to B. cinerea, with potential implications for the screening of grey mould disease‐resistant apple varieties in breeding programmes.     

Hsp104 is required for tolerance to many forms of stress.

Heat-shock proteins (hsps) are induced by many types of stress. In Saccharomyces cerevisiae, a mutation in the HSP104 gene, a member of the highly conserved hsp100 gene family, reduces the ability of log-phase fermenting cells to withstand high temperatures after mild, conditioning pretreatments. Here, we examine the expression of hsp104 and its importance for survival under many different conditions. Hsp104 is expressed at a higher level in respiring cells than in fermenting cells and is required for the unusually high basal thermotolerance of respiring cells. Its expression in stationary phase cells and spores is crucial for the naturally high thermotolerance of these cell types and for their long-term viability at low temperatures. The protein is of critical importance in tolerance to ethanol and of moderate importance in tolerance to sodium arsenite. Thus, the hsp104 mutation establishes the validity of a long-standing hypothesis in the heat-shock field, namely, that hsps have broadly protective functions. Further, that a single protein is responsible for tolerance to heat, ethanol, arsenite and long-term storage in the cold indicates that the underlying causes of lethality are similar in an extraordinary variety of circumstances. Finally, the protein is of little or no importance in tolerance to copper and cadmium, suggesting that the lethal lesions produced by these agents are fundamentally different from those produced by heat.     

The Arabidopsis LECTIN RECEPTOR KINASE-VI.2 is a functional protein kinase and is dispensable for basal resistance to Botrytis cinerea

Sensing of microbial pathogens by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) elicits a defense program known as PAMP-triggered immunity (PTI). Recently, we have shown that the Arabidopsis thaliana L-TYPE LECTIN RECEPTOR KINASE-VI.2 (LecRK-VI.2) positively regulates bacterial PTI. In this report, we suggest by in silico analysis that the kinase domain of LecRK-VI.2 is functional. LecRK-VI.2 also demonstrated auto-phosphorylation activity in vitro in the presence of divalent metal cations indicating that LecRK-VI.2 has the ability to auto-phosphorylate. We further investigate the role of LecRK-VI.2 in Arabidopsis resistance to the necrotrophic fungal pathogen Botrytis cinerea. Disruption of LecRK-VI.2 did not affect Arabidopsis resistance to B. cinerea. Accordingly, wild-type upregulation levels of PTI-responsive WRKY53, FRK1, NHL10, CYP81F2 and CBP60 g after treatment with the fungal PAMP chitin were observed in lecrk-VI.2-1. These data provide evidences that the kinase domain of LecRK-VI.2 is active and show that LecRK-VI.2 is not critical for resistance to the fungal pathogen B. cinerea.