Role of the penetration‐resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race‐specific resistance in Arabidopsis thaliana

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host‐encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall‐based defense against the non‐adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run‐away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall‐based defense against non‐host pathogens.     

Powdery mildew suppresses herbivore‐induced plant volatiles and interferes with parasitoid attraction in Brassica rapa

The co‐occurrence of different antagonists on a plant can greatly affect infochemicals with ecological consequences for higher trophic levels. Here we investigated how the presence of a plant pathogen, the powdery mildew Erysiphe cruciferarum, on Brassica rapa affects (1) plant volatiles emitted in response to damage by a specialist herbivore, Pieris brassicae; (2) the attraction of the parasitic wasp Cotesia glomerata and (3) the performance of P. brassicae and C. glomerata. Plant volatiles were significantly induced by herbivory in both healthy and mildew‐infected plants, but were quantitatively 41% lower for mildew‐infected plants compared to healthy plants. Parasitoids strongly preferred Pieris‐infested plants to dually‐infested (Pieris + mildew) plants, and preferred dually infested plants over only mildew‐infected plants. The performance of P. brassicae was unaffected by powdery mildew, but C. glomerata cocoon mass was reduced when parasitized caterpillars developed on mildew‐infected plants. Thus, avoidance of mildew‐infested plants may be adaptive for C. glomerata parasitoids, whereas P. brassicae caterpillars may suffer less parasitism on mildew‐infected plants in nature. From a pest management standpoint, the concurrent presence of multiple plant antagonists can affect the efficiency of specific natural enemies, which may in turn have a negative impact on the regulation of pest populations.     

mlo‐based powdery mildew resistance in hexaploid bread wheat generated by a non‐transgenic TILLING approach

Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non‐transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss‐of‐function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss‐of‐function alleles (mlo) of barley Mlo are known to confer durable broad‐spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo‐A1, TaMlo‐B1 and TaMlo‐D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non‐conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non‐transgenic, powdery mildew‐resistant bread wheat varieties.     

Interaction of a Blumeria graminis f. sp. hordei effector candidate with a barley ARF‐GAP suggests that host vesicle trafficking is a fungal pathogenicity target

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1–BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana‐infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non‐adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin‐conjugating enzyme, and an ADP ribosylation factor‐GTPase‐activating protein (ARF‐GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF‐GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF‐GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence‐associated host vesicle trafficking.     

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [¹⁴C]‐acetyl‐CoA to oligogalacturonides. Through site‐directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.     

Overexpression of FBR41 enhances resistance to sphinganine analog mycotoxin‐induced cell death and Alternaria stem canker in tomato

Fumonisin B1 (FB1) and Alternaria alternate f. sp. lycopersici (AAL)‐toxin are classified as sphinganine analog mycotoxins (SAMTs), which induce programmed cell death (PCD) in plants and pose health threat to humans who consume the contaminated crop products. Herein, Fumonisin B1 Resistant41 (FBR41), a dominant mutant allele, was identified by map‐based cloning of Arabidopsis FB1‐resistant mutant fbr41, then ectopically expressed in AAL‐toxin sensitive tomato (Solanum lycopersicum) cultivar. FBR41‐overexpressing tomato plants exhibited less severe cell death phenotype upon AAL‐toxin treatment. Analysis of free sphingoid bases showed that both fbr41 and FBR41‐overexpressing tomato plants accumulated less sphinganine and phytosphingosine upon FB1 and AAL‐toxin treatment, respectively. Alternaria stem canker is a disease caused by AAL and responsible for severe economic losses in tomato production, and FBR41‐overexpressing tomato plants exhibited enhanced resistance to AAL with decreased fungal biomass and less cell death, which was accompanied by attenuated accumulation of free sphingoid bases and jasmonate (JA). Taken together, our results indicate that FBR41 is potential in inhibiting SAMT‐induced PCD and controlling Alternaria stem canker in tomato.     

Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat

Wheat (Triticum aestivum L.) incurs significant yield losses from powdery mildew, a major fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). enhanced disease resistance1 (EDR1) plays a negative role in the defense response against powdery mildew in Arabidopsis thaliana; however, the edr1 mutant does not show constitutively activated defense responses. This makes EDR1 an ideal target for approaches using new genome‐editing tools to improve resistance to powdery mildew. We cloned TaEDR1 from hexaploid wheat and found high similarity among the three homoeologs of EDR1. Knock‐down of TaEDR1 by virus‐induced gene silencing or RNA interference enhanced resistance to powdery mildew, indicating that TaEDR1 negatively regulates powdery mildew resistance in wheat. We used CRISPR/Cas9 technology to generate Taedr1 wheat plants by simultaneous modification of the three homoeologs of wheat EDR1. No off‐target mutations were detected in the Taedr1 mutant plants. The Taedr1 plants were resistant to powdery mildew and did not show mildew‐induced cell death. Our study represents the successful generation of a potentially valuable trait using genome‐editing technology in wheat and provides germplasm for disease resistance breeding.     

De novo indol-3-ylmethyl glucosinolate biosynthesis,and not long-distance transport,contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

RPN1a, a 26S proteasome subunit, is required for innate immunity in Arabidopsis

Accumulating evidence shows that proper degradation of proteins that affect defense responses in a positive or negative manner is critical in plant immunity. However, the role of plant degradation systems such as the 26S proteasome in plant immunity is not well understood. Loss‐of‐function mutations in EDR2 (ENHANCED DISEASE RESISTANCE 2) lead to increased resistance to the adapted biotrophic powdery mildew pathogen Golovinomyces cichoracearum. To study the molecular interactions between powdery mildew pathogen and Arabidopsis, we performed a screen for suppressors of edr2 and found that mutation in the gene that encodes RPN1a, a subunit of the 26S proteasome, suppressed edr2‐associated disease resistance phenotypes. In addition, RPN1a is required for edr1‐ and pmr4‐mediated powdery mildew resistance and mildew‐induced cell death. Furthermore, we show that rpn1a displayed enhanced susceptibility to the fungal pathogen G. cichoracearum and to virulent and avirulent bacterial Pto DC3000 strains, which indicated that rpn1a has defects in basal defense and resistance (R) protein‐mediated defense. RPN1a–GFP localizes to both the nucleus and cytoplasm. Accumulation of RPN1a is affected by salicylic acid (SA) and the rpn1a mutant has defects in SA accumulation upon Pto DC3000 infection. Further analysis revealed that two other subunits of the 26S proteasome, RPT2a and RPN8a are also involved in edr2‐mediated disease resistance. Based on these results, we conclude that RPN1a is required for basal defense and R protein‐mediated defense. Our data provide evidence that some subunits of the 26S proteasome are involved in innate immunity in Arabidopsis.     

Tryptophan‐derived secondary metabolites in Arabidopsis thaliana confer non‐host resistance to necrotrophic Plectosphaerella cucumerina fungi

A defence pathway contributing to non‐host resistance to biotrophic fungi in Arabidopsis involves the synthesis and targeted delivery of the tryptophan (trp)‐derived metabolites indol glucosinolates (IGs) and camalexin at pathogen contact sites. We have examined whether these metabolites are also rate‐limiting for colonization by necrotrophic fungi. Inoculation of Arabidopsis with adapted or non‐adapted isolates of the ascomycete Plectosphaerella cucumerina triggers the accumulation of trp‐derived metabolites. We found that their depletion in cyp79B2 cyp79B3 mutants renders Arabidopsis fully susceptible to each of three tested non‐adapted P. cucumerina isolates, and super‐susceptible to an adapted P. cucumerina isolate. This assigns a key role to trp‐derived secondary metabolites in limiting the growth of both non‐adapted and adapted necrotrophic fungi. However, 4‐methoxy‐indol‐3‐ylmethylglucosinolate, which is generated by the P450 monooxygenase CYP81F2, and hydrolyzed by PEN2 myrosinase, together with the antimicrobial camalexin play a minor role in restricting the growth of the non‐adapted necrotrophs. This contrasts with a major role of these two trp‐derived phytochemicals in limiting invasive growth of non‐adapted biotrophic powdery mildew fungi, thereby implying the existence of other unknown trp‐derived metabolites in resistance responses to non‐adapted necrotrophic P. cucumerina. Impaired defence to non‐adapted P. cucumerina, but not to the non‐adapted biotrophic fungus Erysiphe pisi, on cyp79B2 cyp79B3 plants is largely restored in the irx1 background, which shows a constitutive accumulation of antimicrobial peptides. Our findings imply differential contributions of antimicrobials in non‐host resistance to necrotrophic and biotrophic pathogens.     

Identification and Analysis of Powdery Mildew‐Responsive miRNAs in Wheat

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most consistently damaging diseases of common wheat worldwide and greatly affects crop productivity. Recently, several plant microRNAs (miRNAs) have been reported as gene expression regulators related to various adverse environments. However, up to now, less is known on the roles of miRNAs in powdery mildew infection response of wheat. In this study, miRNA expression patterns were investigated for identifying Bgt‐responsive miRNAs in wheat leaves using a plant miRNA microarray platform. A total of 79 miRNAs from 24 families were detected in wheat leaves. Among those, seven miRNAs were further validated to be involved in wheat powdery mildew response and two of them have never been reported. In addition, their target expression profiles showed a negative correlation with that of the seven miRNAs in mock‐ and Bgt‐infected samples furtherly proved, which in turn as the robust evidence, that those seven powdery mildew‐responsive miRNAs are highly reliable. These findings could extend the current view about miRNAs as ubiquitous regulators under stress conditions.     

Nucleocytoplasmic trafficking is essential for BAK1‐ and BKK1‐mediated cell‐death control

BRI1‐ASSOCIATED KINASE 1 (BAK1) was initially identified as a co‐receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1‐LIKE 1 (BKK1) regulate a BR‐independent cell‐death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid‐ and light‐dependent cell‐death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1–1). Genetic analyses indicated that cell‐death symptoms in a weak double mutant, bak1–3 bkk1–1, were completely suppressed by the loss‐of‐function mutation in SBB1, which encodes a nucleoporin (NUP) 85‐like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1‐located sub‐complex was also able to rescue the cell‐death phenotype of bak1–3 bkk1–1. In addition, a DEAD‐box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell‐death symptoms of bak1–3 bkk1–1. Further analyses indicated that export of poly(A)⁺ RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over‐expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell‐death phenotype of bak1–3 bkk1–1. Mutants suppressing cell‐death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1‐ and BKK1‐mediated cell‐death control.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.