Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea ( Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus ( Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.     

A mutation in the GTP hydrolysis site of Arabidopsis dynamin-related protein 1E confers enhanced cell death in response to powdery mildew infection

We screened for mutants of Arabidopsis thaliana that displayed enhanced disease resistance to the powdery mildew pathogen Erysiphe cichoracearum and identified the edr3 mutant, which formed large gray lesions upon infection with E. cichoracearum and supported very little sporulation. The edr3-mediated disease resistance and cell death phenotypes were dependent on salicylic acid signaling, but independent of ethylene and jasmonic acid signaling. In addition, edr3 plants displayed enhanced susceptibility to the necrotrophic fungal pathogen Botrytis cinerea, but showed normal responses to virulent and avirulent strains of Pseudomonas syringae pv. tomato. The EDR3 gene was isolated by positional cloning and found to encode Arabidopsis dynamin-related protein 1E (DRP1E). The edr3 mutation caused an amino acid substitution in the GTPase domain of DRP1E (proline 77 to leucine) that is predicted to block GTP hydrolysis, but not GTP binding. A T-DNA insertion allele in DRP1E did not cause powdery mildew-induced lesions, suggesting that this phenotype is caused by DRP1E being locked in the GTP-bound state, rather than by a loss of DRP1E activity. Analysis of DRP1E-green fluorescent protein fusion proteins revealed that DRP1E is at least partially localized to mitochondria. These observations suggest a mechanistic link between salicylic acid signaling, mitochondria and programmed cell death in plants.     

Differential expression of putative cell death regulator genes in near-isogenic,resistant and susceptible barley lines during interaction with the powdery mildew fungus

We analysed pathogenesis-related expression of genes, that are assumed to be involved in ubiquitous plant defence mechanisms like the oxidative burst, the hypersensitive cell death reaction (HR) and formation of localized cell wall appositions (papillae). We carried out comparative northern blot and RT-PCR studies with near-isogenic barley (Hordeum vulgareL. cv. Pallas) lines (NILs) resistant or susceptible to the powdery mildew fungus race A6 (Blumeria graminis f.sp. hordei, BghA6). The NILs carrying one of the R-genes Mla12, Mlg or the mlo mutant allele mlo5 arrest fungal development by cell wall appositions (mlo5) or a HR (Mla12) or both (Mlg). Expression of an aspartate protease gene, an ascorbate peroxidase gene and a newly identified cysteine protease gene was up-regulated after inoculation with BghA6, whereas the constitutive expression-level of a BAS gene, that encodes an alkyl hydroperoxide reductase, was reduced. Expression of a newly identified barley homologue of a mammalian cell death regulator, Bax inhibitor 1, was enhanced after powdery mildew inoculation. An oxalate oxidase-like protein was stronger expressed in NILS expressing penetration resistance. A so far unknown gene that putatively encodes the large subunit of a superoxide generating NADPH oxidases was constitutively expressed in barley leaves and its expression pattern did not change after inoculation. A newly identified barley Rac1 homologue was expressed constitutively, such as the functionally linked NADPH oxidase gene. Gene expression patterns are discussed with regard to defence mechanisms and signal transduction.     

Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus

Localized cell wall modification and accumulation of antimicrobial compounds beneath sites of fungal attack are common mechanisms for plant resistance to fungal penetration. In barley (Hordeum vulgare) leaves, light-microscopically visible vesicle-like bodies (VLBs) containing H(2)O(2) or phenolics frequently accumulate around cell wall appositions (syn. papillae), in which the penetration attempt of the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) is halted. By ultrastructural analyses, we demonstrated that the Bgh-induced VLBs represent different structures. VLBs intensively stained by H(2)O(2)-reactive dyes were actually small papillae instead of cytoplasmic vesicles. Other VLBs were identified as osmiophilic bodies or multivesicular compartments, designated paramural bodies (PMBs) and multivesicular bodies (MVBs). MVBs seemingly followed two distinct pathways: either they were engulfed by the tonoplast for degradation in the vacuole or they fused with the plasma membrane to release their internal vesicles into the paramural space and hence could be the origin of PMBs. MVBs and PMBs appeared to be multicomponent kits possibly containing building blocks to be readily assembled into papilla and antimicrobial compounds to be discharged against fungal penetration. Finally, we propose that released paramural vesicles might be similar to exosomes in animal cells.     

ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis

The molecular interactions between Arabidopsis and the pathogenic powdery mildew Golovinomyces cichoracearum were studied by characterizing a disease-resistant Arabidopsis mutant atg2-2. The atg2-2 mutant showed enhanced resistance to powdery mildew and dramatic mildew-induced cell death as well as early senescence phenotypes in the absence of pathogens. Defense-related genes were constitutively activated in atg2-2. In atg2-2 mutants, spontaneous cell death, early senescence and disease resistance required the salicylic acid (SA) pathway, but interestingly, mildew-induced cell death was not fully suppressed by inactivation of SA signaling. Thus, cell death could be uncoupled from disease resistance, suggesting that cell death is not sufficient for resistance to powdery mildew. ATG2 encodes autophagy-related 2, a protein known to be involved in the early steps of autophagosome biogenesis. The atg2-2 mutant exhibited typical autophagy defects in autophagosome formation. Furthermore, mutations in several other ATG genes, including ATG5, ATG7 and ATG10, exhibited similar powdery mildew resistance and mildew-induced cell death phenotypes. Taken together, our findings provide insights into the role of autophagy in cell death and disease resistance, and may indicate general links between autophagy, senescence, programmed cell death and defense responses in plants.     

Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus

Small monomeric G-proteins of the plant ras (rat sarcome oncogene product) related C3 botulinum toxin substrate (RAC)/Rho of plants (ROP) family are molecular switches in signal transduction of many cellular processes. RAC/ROPs regulate hormone effects, subcellular gradients of Ca2+, the organisation of the actin cytoskeleton and the production of reactive oxygen intermediates. Therefore, we followed a genetic bottom-up strategy to study the role of these proteins during the interaction of barley (Hordeum vulgare L.) with the fungal biotrophic pathogen Blumeria graminis f.sp. hordei (Bgh). We identified six barley RAC/ROP proteins and studied their gene expression. Five out of six Rac/Rop genes were expressed constitutively in the leaf epidermis, which is the site of interaction with Bgh. None of the genes showed enhancement of mRNA abundance after inoculation with Bgh. After microprojectile mediated transformation of single barley epidermal cells with constitutively activated mutant RAC/ROP proteins, we found an RAC/ROP-specific enhancement of pathogen accessibility, tagging HvRACB, HvRAC3 and HvROP6 as host proteins potentially involved in the establishment of susceptibility to Bgh. Confocal laser scanning microscopy (CLSM) of green fluorescent protein (GFP):HvRAC/ROP-transformed cells revealed varying strengths of plasma membrane association of barley RAC/ROPs. The C-terminal CAAX motif for presumable prenylation or the C-terminal hypervariable region (HVR), respectively, were required for membrane association of the RAC/ROPs. Proper intracellular localisation was essential for HvRACB and HvRAC3 function. Together, our data support the view that different paths of host signal transduction via RAC/ROP G-proteins are involved in processes supporting parasitic entry into epidermal host cells.     

The induction of systemic resistance in barley to powdery mildew infection using salicylates and various phenolic acids

Treatment of the first leaves of barley seedlings with either 5, 10, 15 or 20 mM salicylic acid, sodium salicylate or acetylsalicylic acid resulted in significant reductions in powdery mildew infection on the upper, second leaves. In general, the greatest reduction in mildew infection on the second leaves was obtained by spraying the first leaves with a 15 mM concentration of the compounds. Although the largest reduction in mildew infection of the upper leaves was obtained when the compounds were applied to the first leaves 1–2 days before inoculation, very substantial reductions in infection were still obtained if the first leaves were treated 12 days before inoculation. The three compounds had little direct effect on mildew infection. When ¹⁴C-salicylic acid was fed to first leaves of barley seedlings, uptake was rapid and increased with time. Within 6 h, 0.2% of the salicylic acid appeared in the second leaf and by 24 h after feeding, this had increased to 1.4% (1.1 μmol salicylic acid g^-1 fresh wt). The application of various phenolic acids to first leaves also led to reductions in mildew infection on the second leaves. In particular, treatment of the first leaves with 1 mM vanillic acid, isovanillic acid or syringic acid, reduced mildew infection of the second leaves by 81–87%.     

Differences in early callose deposition during adapted and non-adapted powdery mildew infection of resistant Arabidopsis lines

The deposition of callose, a (1,3)-β-glucan cell wall polymer, can play an essential role in the defense response to invading pathogens. We could recently show that Arabidopsis thaliana lines with an overexpression of the callose synthase gene PMR4 gained complete penetration resistance to the adapted powdery mildew Golovinomyces cichoracearum and the non-adapted powdery mildew Blumeria graminis f. sp hordei. The penetration resistance is based on the transport of the callose synthase PMR4 to the site of attempted fungal penetration and the subsequent formation of enlarged callose deposits. The deposits differed in their total diameter comparing both types of powdery mildew infection. In this study, further characterization of these callose deposits revealed that size differences were especially pronounced in the core region of the deposits. This suggests that specific, pathogen-dependent factors exist, which might regulate callose synthase transport to the core region of forming deposits.     

A novel strain of Pseudozyma aphidis from mulberry parasitises the conidia of mulberry powdery mildew fungus Phyllactinia sp. and its biocontrol effect in the fields

Plant surface is colonised with a vast community of non-pathogenic epiphytic microorganisms which play an important role in host defence. In the present study, we reported a fungus from mulberry leaf surface that showed an antagonistic effect against mulberry powdery mildew fungal pathogen Phyllactinia sp. This novel isolate is a yeast-like fungus that was identified as Pseudozyma aphidis CNm2012 based on morphologic and phylogenetic analysis. According to our research, P. aphidis CNm2012 directly acted on the powdery mildew conidia via parasitism which caused conidial atrophy, collapse and eventually, cleavage and death. During the parasitic process, we found the isolate could gather around the conidia of Phyllactinia sp. and form hyphae that grew on the conidial surface and utilise the conidia as a nutrient source. Field studies revealed that P. aphidis CNm2012 could suppress the disease incidence of mulberry powdery mildew caused by Phyllactinia sp., and reduce the disease severity. To our knowledge, it is the first report of P. aphidis directly against powdery mildew fungus Phyllactinia spp. by parasitism. Our results indicated that P. aphidis CNm2012 could be served as an environmentally friendly alternative of chemical pesticides to manage mulberry powdery mildew disease.     

Seedling resistance of wheat to the powdery mildew fungus,Erysiphe graminis f. sp. tritici. I. Resistance of first leaves

During primary infection by conidia ofErysiphe graminis f. sp.tritici, three mechanisms of resistance operate in first leaves of 8-day-old seedlings of both resistant and susceptible wheats. The first mechanism, operating at the penetration site, is responsible for the failure of penetrations attempted by primary germ tubes (PGT). The second mechanism is concerned with the abortion of haustoria in normal-appearing host cells. The third mechanism relates to the abortion of haustoria and the hypersensitivity of the penetrated host cells.With the inoculum-level of 19–24 conidia/mm², the three mechanisms together prevented 89.3 % of the attempted penetrations by PGT from producing normal haustoria in resistant wheat Purdue 5752C1-7-5-1 and 37.4 % in the susceptible wheat Vermillion. The first mechanism accounted for the prevention of 73.3 % of the attempted PGT penetrations on Purdue 5752C1-7-5-1 and 36 % on Vermillion. The second mechanism was responsible for stopping 19 % of all the successful penetrations in Purdue 5752C1-7-5-1 and 0.8 % in Vermillion. The third mechanism accounted for the failure of 41 % of all the successful penetrations in Purdue 5752C1-7-5-1 and 1.4% in Vermillion. Thirty-six hours after inoculation, 10.7% of all the attempted PGT penetrations appeared to be developing normally in first leaves of 8-day-old seedlings of resistant wheat Purdue 5752C1-7-5-1 as compared to 62.6 % in the susceptible wheat Vermillion.This appears to be the first report showing the relative effectiveness of various mechanisms of resistance concerning any powdery mildew fungus.     

The effects of host carbon dioxide, nitrogen and water supply on the infection of wheat by powdery mildew and aphids

In two experiments, winter wheat (Triticum aestivum cv. Cerco) was grown in 350 (ambient) and 700 μmol mol^-1 (elevated) atmospheric CO₂ concentrations. In the first experiment, plants were grown at five levels of nitrogen fertilization, and in the second experiment, plants were grown at three levels of water supply. All plants were infected with powdery mildew, caused by the fungus Erysiphe graminis. Plants grown in elevated atmospheric CO₂ concentrations had significantly reduced % shoot nitrogen contents and significantly increased % shoot water contents. At elevated atmospheric CO₂ concentrations, where plant nitrogen content was significantly reduced, the severity of mildew infection was significantly reduced, and where host water content was significantly increased, the severity of mildew infection was significantly increased. In a moderate water supply treatment, the plants grown in elevated atmospheric CO₂ concentrations had significantly reduced nitrogen contents (9·9%) and significantly increased water content (4%), the amount of mildew infection was unchanged. The severity of mildew infection appeared to be more sensitive to host water content than to host nitrogen content.     

Regulation of plant defense responses in Arabidopsis by EDR2, a PH and START domain-containing protein

We have identified an Arabidopsis mutant that displays enhanced disease resistance (edr2) to the biotrophic powdery mildew pathogen Erysiphe cichoracearum. Inhibition of fungal growth on edr2 mutant leaves occurred at a late stage of the infection process and coincided with formation of necrotic lesions approximately 5 days after inoculation. Double-mutant analysis revealed that edr2-mediated resistance is suppressed by mutations that inhibit salicylic acid (SA)-induced defense signaling, including npr1, pad4 and sid2, demonstrating that edr2-mediated disease resistance is dependent on SA. However, edr2 showed normal responses to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000. EDR2 appears to be constitutively transcribed in all tissues and organs and encodes a novel protein, consisting of a putative pleckstrin homology (PH) domain and a steroidogenic acute regulatory protein-related lipid-transfer (START) domain, and contains an N-terminal mitochondrial targeting sequence. The PH and START domains are implicated in lipid binding, suggesting that EDR2 may provide a link between lipid signaling and activation of programmed cell death mediated by mitochondria.     

玉米中抗病基因myb1和NDR1同源序列的荧光原位杂交物理定位

以烟草和拟南芥中的单拷贝抗病基因ｍｙｂ１和ＮＤＲ１作探针,利用荧光原位杂交的方法分别对这两个基因在玉米（Ｚｅａ ｍａｙｓ Ｌ．）和烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）、玉米和拟南芥（Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）中的同源性做了研究。杂交结果表明ｍｙｂ１和ＮＤＲ１的同源序列分别位于玉米第８、５染色体,单个信号位置表明０这两个基因的同源序列在玉米基因组中只有     

Physical Mapping of the Sequences Homologous to Disease Resistance Genes myb1 and NDR1 in Maize

Using fluorescence in situ hybridization, the authors investigated the homology between three plant species, maize (Zea mays L.) and tobacco (Nicotiana tabacum L.), maize and Arabidopsis thaliana (L.) Heynh. at cytogenetic level using two probes corresponding to functional disease resistance genes myb1 and NDR1 in Arabidopsis and tobacco respectively. The hybridization signals of the tested probes were detected in maize chromosomes 8 and 5 respectively, and the single location of each of the two probes showed only single copy of them in maize genome. The results provided a valuable insight into searching for genes associated with programmed cell death in plants using heterologous probe with comparative genetic approach. In addition, the improvements of FISH technique using heterologous probes were discussed.