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.     

Suppression of edr2-mediated powdery mildew resistance, cell death and ethylene-induced senescence by mutations in ALD1 in Arabidopsis

EDR2 is a negative regulator of the defense response and cell death in Arabidopsis. Loss-of-function of EDR2 leads to enhanced resistance to powdery mildew. To identify new components in the EDR2 signal transduction pathway, mutations that suppress edr2 resistant phenotypes were screened. Three mutants, edts5-1, edts5-2 and edts5-3 (edr (t)wo (s)uppressor 5), were identified. The EDTS5 gene was identified by map-based cloning and previously was shown to encode an aminotransferase (ALD1). Therefore we renamed these three alleles ald1-10, ald1-11 and ald1-12, respectively. Mutations in ALD1 suppressed all edr2-mediated phenotypes, including powdery mildew resistance, programmed cell death and ethylene-induced senescence. Accumulation of hydrogen peroxide in edr2 was also suppressed by ald1 mutation. The expression of defense-related genes was up-regulated in the edr2 mutant, and the up-regulation of those genes in edr2 was suppressed in the edr2/ald1 double mutant. The ald1 single mutant displayed delayed ethylene-induced senescence. In addition, ald1 mutation suppressed edr1-mediated powdery mildew resistance, but could not suppress the edr1/edr2 double-mutant phenotype. These data demonstrate that ALD1 plays important roles in edr2-mediated defense responses and senescence, and revealed a crosstalk between ethylene and salicylic acid signaling mediated by ALD1 and EDR2.     

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.     

ENHANCED DISEASE RESISTANCE4 Associates with CLATHRIN HEAVY CHAIN2 and Modulates Plant Immunity by Regulating Relocation of EDR1 in Arabidopsis

Obligate biotrophs, such as the powdery mildew pathogens, deliver effectors to the host cell and obtain nutrients from the infection site. The interface between the plant host and the biotrophic pathogen thus represents a major battleground for plant-pathogen interactions. Increasing evidence shows that cellular trafficking plays an important role in plant immunity. Here, we report that Arabidopsis thaliana ENHANCED DISEASE RESISTANCE4 (EDR4) plays a negative role in resistance to powdery mildew and that the enhanced disease resistance in edr4 mutants requires salicylic acid signaling. EDR4 mainly localizes to the plasma membrane and endosomal compartments. Genetic analyses show that EDR4 and EDR1 function in the same genetic pathway. EDR1 and EDR4 accumulate at the penetration site of powdery mildew infection, and EDR4 physically interacts with EDR1, recruiting EDR1 to the fungal penetration site. In addition, EDR4 interacts with CLATHRIN HEAVY CHAIN2 (CHC2), and edr4 mutants show reduced endocytosis rates. Taken together, our data indicate that EDR4 associates with CHC2 and modulates plant immunity by regulating the relocation of EDR1 in Arabidopsis.     

The THO/TREX complex functions in disease resistance in Arabidopsis

Powdery mildew pathogens are biotrophic fungi that infect large number of plant species. EDR1 (ENHANCED DISEASE RESISTANCE 1) is a negative regulator of plant disease resistance, and loss-of-function in the EDR1 gene confers enhanced disease resistance to powdery mildew pathogen Golovinomyces cichoracearum. In an edr1 suppressor screen, we recently found that a mutation in HPR1, a component of the THO/TREX complex, suppresses edr1-mediated disease resistance, however the hpr1 mutation enhances the ethylene-induced senescence in edr1. The hpr1 single mutant displays enhanced susceptibility, indicating that HPR1 is involved in plant defense responses.¹ THO/TREX is a conserved protein complex that functions in pre-mRNA processing and mRNA export. Several components of THO/TREX complex in Arabidopsis have been identified. By searching Arabidopsis database, we found that Arabidopsis (Columbia-0) has two copies of UAP56, another component of the THO/TREX complex, and the UAP56 proteins are highly conserved. Similar to human UAP56 protein, Arabidopsis UAP56 also localizes to the nucleus, showing a pattern similar to the splicing speckles. Further characterization of the components of THO/TREX in Arabidopsis will provide new insights into the role of THO/TREX in defense responses in plants.     

Regulation of plant disease resistance, stress responses, cell death, and ethylene signaling in Arabidopsis by the EDR1 protein kinase

ENHANCED DISEASE RESISTANCE 1 (EDR1) encodes a CTR1-like kinase and was previously reported to function as a negative regulator of disease resistance and ethylene-induced senescence. Here, we report that the edr1 mutant displays enhanced stress responses and spontaneous necrotic lesions under drought conditions in the absence of pathogen, suggesting that EDR1 is also involved in stress response signaling and cell death regulation. Double mutant analysis revealed that these drought-induced phenotypes require salicylic acid but not ethylene signaling pathways. In addition, the edr1-mediated ethylene-induced senescence phenotype was suppressed by mutations in EIN2, but not by mutations in SID2, PAD4, EDS1, or NPR1, suggesting that EDR1 functions at a point of cross talk between ethylene and salicylic acid signaling that impinges on senescence and cell death. Two edr1-associated phenotypes, drought-induced growth inhibition and ethylene-induced senescence, were suppressed by mutations in ORE9, implicating ubiquitin-mediated protein degradation in the regulation of these phenotypes. However, the ore9 mutation did not suppress edr1-mediated enhanced disease resistance to powdery mildew or spontaneous lesions, indicating that these phenotypes are controlled by separate signaling pathways. To investigate the function of the EDR1 kinase domain, we expressed the C-terminal third of EDR1 in wild-type Columbia and edr1 backgrounds under the control of a dexamethasone-inducible promoter. Overexpression of the EDR1 kinase domain in an edr1 background had no obvious effect on edr1-associated phenotypes. However, overexpression of the EDR1 kinase domain in a wild-type Columbia background caused dominant negative phenotypes, including enhanced disease resistance to powdery mildew and enhanced ethylene-induced senescence; thus, the overexpressed EDR1 kinase domain alone does not exert EDR1 function, but rather negatively affects the function of native EDR1 protein.     

An Arabidopsis mutant with enhanced resistance to powdery mildew.

We have identified an Arabidopsis mutant that displays enhanced disease resistance to the fungus Erysiphe cichoracearum, causal agent of powdery mildew. The edr1 mutant does not constitutively express the pathogenesis-related genes PR-1, BGL2, or PR-5 and thus differs from previously described disease-resistant mutants of Arabidopsis. E. cichoracearum conidia (asexual spores) germinated normally and formed extensive hyphae on edr1 plants, indicating that the initial stages of infection were not inhibited. Production of conidiophores on edr1 plants, however, was <16% of that observed on wild-type Arabidopsis. Reduction in sporulation correlated with a more rapid induction of defense responses. Autofluorescent compounds and callose accumulated in edr1 leaves 3 days after inoculation with E. cichoracearum, and dead mesophyll cells accumulated in edr1 leaves starting 5 days after inoculation. Macroscopic patches of dead cells appeared 6 days after inoculation. This resistance phenotype is similar to that conferred by "late-acting" powdery mildew resistance genes of wheat and barley. The edr1 mutation is recessive and maps to chromosome 1 between molecular markers ATEAT1 and NCC1. We speculate that the edr1 mutation derepresses multiple defense responses, making them more easily induced by virulent pathogens.     

Variation in Oidium neolycopersici development on host and non-host plant species and their tissue defence responses

Detailed studies of the infection processes of Oidium neolycopersici (tomato powdery mildew) and plant tissue responses were carried out on 10 Lycopersicon spp. accessions and one of each species of Cucumis sativus, Datura stramonium, Lactuca sativa, Petunia hybrida and Pisum sativum with different levels of resistance. Germination of O. neolycopersici conidia was not inhibited by the host plant. However, in the early stages of O. neolycopersici infection significant differences in conidial germ tube development on resistant and susceptible plant lines were observed. The first substantial differences appeared after 24 h post inoculation (hpi). Mycelia and conidiophores developed on susceptible lines; however, there was no mycelial development on resistant plant lines. The most frequent resistant response in Lycopersicon species was the necrotic (hypersensitive) reaction (HR), occasionally followed by pathogen development. The completely resistant accession L. hirsutum (LA 1347) showed only a limited number of necrotic host cells per infection site (2%). In Oidium resistant tomato lines OR 4061 and OR 960008 the existence of adult resistance was detected. This phenomenon occurred mainly in accession OR 4061. Rapid development and profuse sporulation of O. neolycopersici was observed on juvenile plants (6–8 wk old), however this was in contrast to the slow development and sporadic sporulation observed on 4 month old plants. Evidence of posthaustorial resistance was observed in the interaction of O. neolycopersici with non-host species (Lactuca sativa and Pisum sativum.) This was in contrast to Datura stramonium and Petunia hybrida, where development of powdery mildew was delayed at a later stage in the infection cycle. With the exception of Pisum sativum, the necrotic (hypersensitive) response was observed often.     

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.     

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.     

Powdery Mildew Resistance in Tomato by Impairment of SlPMR4 and SlDMR1

Genetic dissection of disease susceptibility in Arabidopsis to powdery and downy mildew has identified multiple susceptibility (S) genes whose impairment results in disease resistance. Although several of these S-genes have been cloned and characterized in more detail it is unknown to which degree their function in disease susceptibility is conserved among different plant species. Moreover, it is unclear whether impairment of such genes has potential in disease resistance breeding due to possible fitness costs associated with impaired alleles. Here we show that the Arabidopsis PMR4 and DMR1, genes encoding a callose synthase and homoserine kinase respectively, have functional orthologs in tomato with respect to their S-gene function. Silencing of both genes using RNAi resulted in resistance to the tomato powdery mildew fungus Oidium neolycopersici. Resistance to O. neolycopersici by SlDMR1 silencing was associated with severely reduced plant growth whereas SlPMR4 silencing was not. SlPMR4 is therefore a suitable candidate gene as target for mutagenesis to obtain alleles that can be deployed in disease resistance breeding of tomato.     

Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (<Emphasis Type="Italic">Erysiphe necator</Emphasis>) and their use in marker-assisted breeding

A limited genetic mapping strategy based on simple sequence repeat (SSR) marker data was used with five grape populations segregating for powdery mildew (Erysiphe necator) resistance in an effort to develop genetic markers from multiple sources and enable the pyramiding of resistance loci. Three populations derived their resistance from Muscadinia rotundifolia ‘Magnolia’. The first population (06708) had 97 progeny and was screened with 137 SSR markers from seven chromosomes (4, 7, 9, 12, 13, 15, and 18) that have been reported to be associated with powdery or downy mildew resistance. A genetic map was constructed using the pseudo-testcross strategy and QTL analysis was carried out. Only markers from chromosome 13 and 18 were mapped in the second (04327) and third (06712) populations, which had 47 and 80 progeny, respectively. Significant QTLs for powdery mildew resistance with overlapping genomic regions were identified for different tissue types (leaf, stem, rachis, and berry) on chromosome 18, which distinguishes the resistance in ‘Magnolia’ from that present in other accessions of M. rotundifolia and controlled by the Run1 gene on chromosome 12. The ‘Magnolia’ resistance locus was termed as Run2.1. Powdery mildew resistance was also mapped in a fourth population (08391), which had 255 progeny and resistance from M. rotundifolia ‘Trayshed’. A locus accounting for 50% of the phenotypic variation mapped to chromosome 18 and was named Run2.2. This locus overlapped the region found in the ‘Magnolia’-based populations, but the allele sizes of the flanking markers were different. ‘Trayshed’ and ‘Magnolia’ shared at least one allele for 68% of the tested markers, but alleles of the other 32% of the markers were not shared indicating that the two M. rotundifolia selections were very different. The last population, 08306 with 42 progeny, derived its resistance from a selection Vitis romanetii C166-043. Genetic mapping discovered a major powdery mildew resistance locus termed Ren4 on chromosome 18, which explained 70% of the phenotypic variation in the same region of chromosome 18 found in the two M. rotundifolia resistant accessions. The mapping results indicate that powdery mildew resistance genes from different backgrounds reside on chromosome 18, and that genetic markers can be used as a powerful tool to pyramid these loci and other powdery mildew resistance loci into a single line.     

Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana

This paper reports on six Arabidopsis accessions that show resistance to a wild isolate of the powdery mildew pathogen, Erysiphe cichoracearum . Resistance at 7 days post-inoculation in these accessions was characterized by limited fungal growth and sporadic development of chlorotic or necrotic lesions at inoculation sites. Three accessions, Wa-1, Kas-1 and SI-0, were highly resistant, while the other accessions permitted some fungal growth and conidiation. Papilla formation was a frequent host response; however, cell death appeared to be neither a rapid nor a common response to infection. To determine the genetic basis of resistance, segregation analyses of progeny from crosses between each of the resistant accessions and Columbia ( gl1 ), which is susceptible to the powdery mildew pathogen, were performed. For all accessions except SI-0, resistance was conferred by a single locus. SI-0 was unique in that two unlinked loci controlled the disease reaction phenotype. In accessions Wa-1, Kas-1, Stw-0 and Su-0, powdery mildew resistance was encoded by a semi-dominant allele. However, susceptibility was dominant to resistance in accessions Te-0 and SI-0. Mapping studies revealed that powdery mildew resistances in Kas-1, Wa-1, Te-0, Su-0 and Stw-0 were controlled by five independent loci. This study suggests that the Arabidopsis powdery mildew disease will be a suitable model system in which to investigate powdery mildew diseases.