Competitive performance of transgenic wheat resistant to powdery mildew

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.     

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.     

Inheritance of partial resistance to powdery mildew in spring wheat

Summary Four spring wheat (Triticum aestivum L.) cultivars exhibiting partial resistance to powdery mildew induced by Erysiphe graminis f.sp. tritici were crossed to a common susceptible cultivar to study the inheritance of resistance. The genetic parameters contributing to resistance were estimated by generation means analyses. Additive gene action was the most important genetic component of variation among generation means in all four crosses. Additive by additive effects were significant in one cross and both additive by additive and additive by dominance effects were significant in another. Dominance effects were not significant. The F2/F3 correlations in three crosses ranged from 0.27 to 0.43. Three additional crosses among resistant cultivars were employed to study the effectiveness of selection in improving resistance. By selecting the most resistant plants from the F2 and evaluating the progenies in the F4, increases in resistance ranging from 21% to 31% were obtained. In all crosses, there was transgressive segregation in both directions indicating that the genes conferring resistance to these cultivars differ and exhibit additive effects.     

Nod factor induces soybean resistance to powdery mildew.

Plants possess highly sensitive perception systems by which microbial signal molecules are recognized. In the Bradyrhizobium-soybean (Glycine max (L.) Merr.) symbiosis, recognition is initiated through exchange of signal molecules, generally flavonoids from soybean and lipo-chitooligosaccharides (Nod factors) from the microsymbiont. Application of the Nod factor Nod Bj-V (C18:1, MeFuc) induced soybean resistance to powdery mildew caused by Microsphaera diffusa. Addition of Nod factor (concentrations ranging from 10(-6) to 10(-10) M) to soybean root systems led to reductions in disease incidence. The lowest disease incidence was caused by Nod factor treatment at 10(-6) M. The effect of Nod factor application on fungal growth and development was measured at 4, 12, 48, and 96 h after inoculation. Colony diameter and number of germ tubes per conidium were decreased by 10(-6) M Nod factor. Phenylalanine ammonia lyase (PAL, EC.4.3.1.1.) is the first enzyme of the phenyl propanoid pathway, and is commonly activated as part of plant responses to disease. Treatment of soybean seedlings with Nod factor, through stem wounds, induced PAL activity; the most rapid increase followed treatment with 10(-6) M Nod factor. These data show that soybean plants are able to detect root applied LCO and respond by increased disease resistance.     

Mapping oligogenic resistance to powdery mildew in mungbean with RFLPs

We have used restriction fragment length polymorphisms (RFLPs) to map genes in mungbean (Vigna radiata) that confer partial resistance to the powdery mildew fungus, Erysiphe polygoni. DNA genotypes for 145 RFLP loci spanning 1570 centimorgans of the mungbean genome were assayed in a population of 58 F₂ plants. This population was derived from a cross between a moderately powdery mildew resistant (VC3980A) and a susceptible (TC1966) mungbean parent. F₃ lines derived from the F₂ plants were assayed in the field for powdery mildew response and the results were compared to the RFLP genotype data, thereby identifying loci associated with powdery mildew response. A total of three genomic regions were found to have an effect on powdery mildew response, together explaining 58% of the total variation. At 65 days after planting, two genomic regions were significantly associated with powdery mildew resistance. For both loci, the allele from VC3890A was associated with increased resistance. At 85 days, a third genomic region was also associated with powdery mildew response. For this locus, the allele from the susceptible parent (TC1966) was the one associated with higher levels of powdery mildew resistance. These results indicate that putative partial resistance loci for powdery mildew in mungbean can be identified with DNA markers, even in a population of modest size analyzed at a single location in a single year.     

Inheritance of adult plant resistance to powdery mildew in swedes

A complete diallel set of crosses, including selfs, was produced from 11 inbred lines of swedes and assessed for adult plant resistance to powdery mildew in a replicated field trial. The plants were inoculated with powdery mildew spores on 23 July 1987. Traces of mildew were visible on 3 August. By 14 September the most susceptible inbred line had 95% of its leaf area infected, whereas the three most resistant lines derived from cvs Bangholm Dima (two lines) and Bangholm Magres (one line) had only 5%. Resistance was controlled by partially recessive genes. There was no evidence of maternal effects. The implications for breeding inbred and F₁ hybrid swede cultivars are discussed.     

Transient silencing of VvCSN5 enhances powdery mildew resistance in grapevine (Vitis vinifera)

As one of the most economically important fruit crops in the world, the grapevine (Vitis vinifera) suffers significant yield losses from various pathogens including powdery mildew caused by Erysiphe necator. In contrast, several wild Chinese grapevines, including Vitis pseudoreticulata accession Baihe-35-1, are highly resistant to powdery mildew pathogens. Here, we identified a grapevine gene CSN5 (COP9 signalosome complex subunit 5), designated VvCSN5, that was differentially expressed between the resistant ‘Baihe-35-1’ and susceptible ‘Thompson Seedless’ during powdery mildew isolate Erysiphe necator NAFU1 infection. Moreover, transient silencing of VvCSN5 in ‘Thompson Seedless’ leaves enhanced resistance to En NAFU1. This resistance manifested in cell wall callose deposition at attempted infection sites and hypersensitive response-like cell death of penetrated epidermal cells. Several defense-related marker genes (VvPR1, VvPR3, VvPAD4, and VvRBOHD) had higher basal expression levels in VvCSN5-silenced leaves. In addition, we found the structure and activity of CSN5 promoters in ‘Thompson Seedless’ and ‘Baihe-35-1’ were different, which may have been behind their different resistances to powdery mildew infection. Taken together, these results implied that grapevine CSN5 plays an important role in the response to powdery mildew infection.

     

Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis

The EDR1 gene of Arabidopsis has previously been reported to encode a Raf-like mitogen-activated protein kinase kinase (MAPKK) kinase, and to function as a negative regulator of disease resistance. A phylogenetic analysis of plant and animal protein kinases revealed, however, that plant Raf-like kinases are more closely related to animal mixed lineage kinases (MLKs) than Raf-like kinases, and are deeply divergent from both classes of animal kinases, making inferences of substrate specificity questionable. We, therefore, assayed the kinase activity of recombinant EDR1 protein in vitro. The EDR1 kinase domain displayed autophosphorylation activity and phosphorylated the common MAP kinase substrate myelin basic protein. The EDR1 kinase domain also phosphorylated a kinase-deficient EDR1 protein, indicating that EDR1 autophosphorylation can occur via an intermolecular mechanism. Overexpression of a kinase-deficient full-length EDR1 gene (35S::dnEDR1) in wild-type Arabidopsis plants caused a dominant negative phenotype, conferring resistance to powdery mildew (Erysiphe cichoracearum) and enhancing ethylene-induced senescence. RNA-gel blot analyses showed that the 35S::dnEDR1 transgene was highly transcribed in transgenic plants. Western blot analysis, however, revealed that neither the wild-type nor mutant EDR1 protein could be detected in these lines, indicating that the dominant negative phenotype may be caused by a translational inhibition mechanism rather than by a protein level effect. Overexpression of orthologous dnEDR1 constructs may provide a novel strategy for controlling powdery mildew disease in crops.     

Biochemical genetics of powdery mildew resistance in pea

Summary A biochemical study on phenolic (total phenols and orthodihydroxy phenols) content and on the activities of phenol oxidizing enzymes (peroxidase and polyphenol oxidase) in pea cultivars resistant and susceptible to powdery mildew infection revealed that the resistant cultivars contained higher levels of phenolics and phenol-oxidizing enzymes than the susceptible ones. A further study of their F₁s, F₂s and backcross progenies suggested a high heritability for all biochemical traits. The correlation coefficients between the biochemical parameters and the disease index were also high. Both additive (d) and dominant () components were found to contribute to the inheritance of these constituents.Associate Professor (Genetics), Department of Basic Sciences     

Mapping resistance to powdery mildew in barley reveals a large-effect nonhost resistance QTL

Key message

Resistance factors against non-adapted powdery mildews were mapped in barley. Some QTLs seem effective only to non-adapted mildews, while others also play a role in defense against the adapted form.The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. We investigated which genetic factors determine the nonhost status of barley (Hordeum vulgare L.) to powdery mildews (Blumeria graminis). We set out to verify whether genes involved in nonhost resistance have a wide effectiveness spectrum, and whether nonhost resistance genes confer resistance to the barley adapted powdery mildew. Two barley lines, SusBgt_SC and SusBgt_DC, with some susceptibility to the wheat powdery mildew B. graminis f.sp. tritici (Bgt) were crossed with cv Vada to generate two mapping populations. Each population was assessed for level of infection against four B. graminis ff.spp, and QTL mapping analyses were performed. Our results demonstrate polygenic inheritance for nonhost resistance, with some QTLs effective only to non-adapted mildews, while others play a role against adapted and non-adapted forms. Histology analyses of nonhost interaction show that most penetration attempts are stopped in association with papillae, and also suggest independent layers of defence at haustorium establishment and conidiophore formation. Nonhost resistance of barley to powdery mildew relies mostly on non-hypersensitive mechanisms. A large-effect nonhost resistance QTL mapped to a 1.4 cM interval is suitable for map-based cloning.

     

Isolation of resistance gene analogues to powdery mildew resistance sequences in hexaploid wheat

This paper reports the characterization of the powdery mildew resistance homologous genes family of Triticum aestivum. Using degenerate primer pair for wheat resistance genes, we have cloned seven 3′ truncated powdery mildew resistance gene homologous fragments Tpc5a, Tp25a, Tp25b, Tp3a5a, Tp3a5b, Tp4b5a and Tp4b5b. These fragments were sequenced. The deduced amino acid sequences showed that six of them have premature stop codons. All these sequences had a very high level of similarity to known Pm resistance genes such as Pm3a, Pm3b, Pm3d and pm3f in hexaploid wheat. By ignoring the stop codons in the sequences, their deduced protein sequences were of coiled-coil (CC)-nucleotide binding site (NBS)-leucine repeat rich (LRR) structure. These results suggest that there are many powdery mildew resistance gene analogues in both resistant and susceptible wheat. Among them, small insertion/deletion events and point mutations can result in the diversity of wheat Pm resistance homologous genes.     

Field resistance of spring barley cultivars to powdery mildew in Lithuania

During vegetative period 2004–2005 powdery mildew (Erysiphe graminis DC. f. sp. hordei Em. Marchal) field resistance of spring barley cultivars was investigated at the Lithuanian Institute of Agriculture. The spring barley genotypes tested were Lithuania-registered cultivars, cultivars from genetic resources collection, and the new cultivars used for initial breeding. In total, 23 resistance genes were present in the 84 cultivars studied. Among mono-genes only mlo and 1-B-53 showed very high resistance. Slight powdery mildew necroses (up to 3 scores) formed on cultivars possessing these genes. The maximal powdery mildew (PM) severity reached a score of 8.5 and the area under disease progress curve (AUDPC) a value of 1216.8. The cultivars ‘Primus’, ‘Astoria’, ‘Power’, ‘Harrington’ and ‘Scarlett’ were the most resistant among the non mlo cultivars. Severity of PM on ‘Primus’ reached a score of 3.5 (3.0 of PM necrosis) in average, the other cultivars were diseased from 4.5 (3.0) to 5.0 (2.0). The AUDPC values for these cultivars except ‘Scarlett’ were the lowest (85.0–145.3) among the other cultivars. The highest contrast in development of the other leaf diseases was between highly resistant and susceptible to PM cultivar groups. The fast development of PM depressed development of the other diseases 4.7 times.     

Gene action for adult-plant resistance to powdery mildew in wheat.

Gene action for adult-plant resistance to powdery mildew was studied using generation mean analyses of parents and of F1, F2, and backcross populations derived from a diallel cross of one susceptible and three adult-plant resistant wheat cultivars. Joint scaling tests showed that an additive-dominance model was sufficient to explain the variability in the expression of adult-plant resistance in one cross, while digenic epistasis was involved in the other five crosses. Additive gene effects were predominant; however, dominance was significant in four crosses, additive x additive interaction was significant in three crosses, additive x dominance interaction was significant in three crosses, and dominance x dominance interaction was significant in one cross. Therefore, selection for adult-plant resistance would likely be most effective in advanced generations derived from crosses among the adult-plant resistant cultivars Redcoat, Houser, and Massey.     

Assessment of partial resistance to powdery mildew in hexaploid wheat genotypes

In the present study the degree of partial resistance (PR) of eleven hexaploid wheat (Triticum aestivum L.) genotypes was evaluated in laboratory (ratio of infection units in stage of second germ tube elongation versus stage of appressorium formation — ESH/App) and field conditions (calculating area under the disease progress curve — AUDPC). Based on the obtained data, genotypes with high degree of PR (Estica, GK Csornoc and Lívia), middle-resistant genotypes (Sana, Mv Vilma and Folio), genotypes with low portion of PR (Barbara, Torysa and Proteinka), and supersensitive genotypes (Renesansa and Am22/99) were differentiated. Both approaches appeared to be suitable for PR measuring with a good discriminating capability between the given genotypes. The results were equivalent in both instances. In addition, a new statistical approach permitting comparison of the obtained data is described.     

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.     

Identification of QTLs for resistance to powdery mildew and SSR markers diagnostic for powdery mildew resistance genes in melon (Cucumis melo L.)

Powdery mildew caused by Podosphaera xanthii is an important foliar disease in melon. To find molecular markers for marker-assisted selection, we constructed a genetic linkage map of melon based on a population of 93 recombinant inbred lines derived from crosses between highly resistant AR 5 and susceptible ‘Earl’s Favourite (Harukei 3)’. The map spans 877 cM and consists of 167 markers, comprising 157 simple sequence repeats (SSRs), 7 sequence characterized amplified region/cleavage amplified polymorphic sequence markers and 3 phenotypic markers segregating into 20 linkage groups. Among them, 37 SSRs and 6 other markers were common to previous maps. Quantitative trait locus (QTL) analysis identified two loci for resistance to powdery mildew. The effects of these QTLs varied depending on strain and plant stage. The percentage of phenotypic variance explained for resistance to the pxA strain was similar between QTLs (R ² = 22–28%). For resistance to pxB strain, the QTL on linkage group (LG) XII was responsible for much more of the variance (41–46%) than that on LG IIA (12–13%). The QTL on LG IIA was located between two SSR markers. Using an independent population, we demonstrated the effectiveness of these markers. This is the first report of universal and effective markers linked to a gene for powdery mildew resistance in melon.     

Both stable and unstable QTLs for resistance to powdery mildew are detected in apple after four years of field assessments

Powdery mildew, caused by the ascomycete fungus Podosphaera leucotricha, is one of the most damaging diseases of apple worldwide. Polygenically determined resistance might contribute to a significant increase of resistance to this disease in new cultivars. A quantitative trait locus (QTL) analysis was performed in an F₁ progeny derived from a cross between the apple cultivar Discovery and the apple hybrid TN10-8. Powdery mildew incidence was assessed during four years (five seasons) in spring and/or autumn in a French local orchard. Seven additive and/or dominant QTLs were detected over the five seasons, with effects (R ²) ranging from 7.5% to 27.4% of the progeny phenotypic variation. Two QTLs, on linkage groups (LGs) 2 and 13, were consistently identified and accounted together from 29% to 37% of the phenotypic variation according to the year of assessment. The other QTLs were identified during one (LGs 1, 14), two (LG10), or three (LGs 8, 17) seasons. Their instability indicated a changing genetic determinism according to the year of assessment, for which several hypotheses may be put forward. The QTLs on LGs 2 and 8 mapped close to clusters of resistance gene analogs (RGAs) and major genes for resistance to mildew or apple scab previously identified. The stable QTLs identified on LGs 2 and 13, together with the strong effect QTL located on LG 8, are of special interest for breeding purposes, especially if combined with other major resistance genes.     

Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa

Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.     

Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1–12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. Received: 2 May 2001 / Accepted: 3 August 2001