Structural polymorphisms within a common powdery mildew effector scaffold as a driver of coevolution with cereal immune receptors.

In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7, and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.

Over-accumulation of chloroplast-nucleus located WHIRLY1 in barley leads to a decrease in growth and an enhanced stress resistance.

WHIRLY1 is a chloroplast-nucleus located DNA/RNA-binding protein with functions in development and stress tolerance. By overexpression of HvWHIRLY1 in barley, one line with a 10-fold and two lines with a 50-fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form. Growth of the plants was shown to be compromised in a WHIRLY1 abundance-dependent manner. Over-accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the light reactions of photosynthesis as well as in carbon fixation. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that in nonstress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance PR gene expression increased indicating that under stress conditions oeW1 are capable to further enhance defense.

Ancient variation of the AvrPm17 gene in powdery mildew limits the effectiveness of the introgressed rye Pm17 resistance gene in wheat.

Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable-a phenomenon that remains poorly understood, as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used quantitative trait locus (QTL) mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of reoccurring gene conversion events and are members of a mildew sublineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sublineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangling complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations become an integral part of introgression breeding to ensure effective and durable resistance in wheat.

Genetic Mapping, Identification, and Characterization of a Candidate Susceptibility Gene for Powdery Mildew in Cannabis sativa.

Powdery mildew (PM) in Cannabis sativa is most frequently caused by the biotrophic fungus Golovinomyces ambrosiae. Based on previously characterized variation in susceptibility to PM, biparental populations were developed by crossing the most resistant cultivar evaluated, 'FL 58', with a susceptible cultivar, 'TJ's CBD'. F1 progeny were evaluated and displayed a range of susceptibility, and two were self-pollinated to generate two F2 populations. In 2021, the F2 populations (n = 706) were inoculated with PM and surveyed for disease severity. In both F2 populations, 25% of the progeny were resistant, while the remaining 75% showed a range of susceptibility. The F2 populations, as well as selected F1 progeny and the parents, were genotyped with a single-nucleotide polymorphism array, and a consensus genetic map was produced. A major effect quantitative trait locus on C. sativa chromosome 1 (Chr01) and other smaller-effect quantitative trait loci (QTL) on four other chromosomes were identified. The most associated marker on Chr01 was located near CsMLO1, a candidate susceptibility gene. Genomic DNA and cDNA sequencing of CsMLO1 revealed a 6.8-kb insertion in FL 58, relative to TJ's CBD, of which 846 bp are typically spliced into the mRNA transcript encoding a premature stop codon. Molecular marker assays were developed using CsMLO1 sequences to distinguish PM-resistant and PM-susceptible genotypes. These data support the hypothesis that a mutated MLO susceptibility gene confers resistance to PM in C. sativa and provides new genetic resources to develop resistant cultivars. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Overexpression of cucumber CYP82D47 enhances resistance to powdery mildew and Fusarium oxysporum f. sp. cucumerinum.

Cytochrome P450s are a large family of protein-encoding genes in plant genomes, many of which have not yet been comprehensively characterized. Here, a novel P450 gene, CYP82D47, was isolated and functionally characterized from cucumber (Cucumis sativus L.). Quantitative real-time reverse-transcription polymerase chain reaction analysis revealed that CYP82D47 expression was triggered by salicylic acid (SA) and ethephon (ETH). Expression analysis revealed a correlation between CYP82D47 transcript levels and plant defense responses against powdery mildew (PM) and Fusarium oxysporum f. sp. cucumerinum (Foc). Although no significant differences were observed in disease resistance between CYP82D47-RNAi and wild-type cucumber, overexpression (OE) of CYP82D47 enhanced PM and Foc resistance in cucumber. Furthermore, the expression levels of SA-related genes (PR1, PR2, PR4, and PR5) increased in CYP82D47-overexpressing plants 7 days post fungal inoculation. The levels of ETH-related genes (EIN3 and EBF2) were similarly upregulated. The observed enhanced resistance was associated with the upregulation of SA/ETH-signaling-dependent defense genes. These findings indicate the crucial role of CYP82D47 in pathogen defense in cucumber. CYP82D47-overexpressing cucumber plants exhibited heightened susceptibility to both diseases. The study results offer important insights that could aid in the development of disease-resistant cucumber cultivars and elucidate the molecular mechanisms associated with the functions of CYP82D47.

Temporal dynamics of N-hydroxypipecolic acid and salicylic acid pathways in the disease response to powdery mildew in wheat.

Wheat (Triticum aestivum) is a major staple crop worldwide, and its yields are significantly threatened by wheat powdery mildew (Blumeria graminis f. sp. tritici). Enhancing disease resistance in wheat is crucial for meeting global food demand. This study investigated the disease response in wheat, focusing on the bioactive small molecules salicylic acid (SA), pipecolic acid (Pip), and N-hydroxypipecolic acid (NHP), to provide new insights for molecular breeding. We found that endogenous levels of SA, Pip, and NHP significantly increased in infected plants, with Pip and NHP levels rising earlier than those of SA. Notably, the rate of increase of NHP was substantially higher than that of SA. The gene expression levels of SARD1 and CBP60g, which are transcription factors for SA, Pip, and NHP biosynthesis, increased significantly during the early stages of infection. We also found that during the later stages of infection, the expression of ALD1, SARD4, and FMO1, which encode enzymes for Pip and NHP biosynthesis, dramatically increased. Additionally, ICS1, which encodes a key enzyme involved in SA biosynthesis, also showed increased expression during the later stages of infection. The temporal changes in ICS1 transcription closely mirrored the behavior of endogenous SA levels, suggesting that the ICS pathway is the primary route for SA biosynthesis in wheat. In conclusion, our results suggest that the early accumulation of Pip and NHP cooperates with SA in the disease response against wheat powdery mildew infection.

Application of bulk segregant RNA-Seq (BSR-Seq) and allele-specific primers to study soybean powdery mildew resistance.

BACKGROUND: Powdery mildew (PM) is one of the important soybean diseases, and host resistance could practically contribute to soybean PM management. To date, only the Rmd locus on chromosome (Chr) 16 was identified through traditional QTL mapping and GWAS, and it remains unclear if the bulk segregant RNA-Seq (BSR-Seq) methodology is feasible to explore additional PM resistance that might exist in other varieties. RESULTS: BSR-Seq was applied to contrast genotypes and gene expressions between the resistant bulk (R bulk) and the susceptible bulk (S bulk), as well as the parents. The ∆(SNP-index) and G' value identified several QTL and significant SNPs/Indels on Chr06, Chr15, and Chr16. Differentially expressed genes (DEGs) located within these QTL were identified using HISAT2 and Kallisto, and allele-specific primers (AS-primers) were designed to validate the accuracy of phenotypic prediction. While the AS-primers on Chr06 or Chr15 cannot distinguish the resistant and susceptible phenotypes, AS-primers on Chr16 exhibited 82% accuracy prediction with an additive effect, similar to the SSR marker Satt431. CONCLUSIONS: Evaluation of additional AS-primers in the linkage disequilibrium (LD) block on Chr16 further confirmed the resistant locus, derived from the resistant parental variety 'Kaohsiung 11' ('KS11'), not only overlaps with the Rmd locus with unique up-regulated LRR genes (Glyma.16G213700 and Glyma.16G215100), but also harbors a down-regulated MLO gene (Glyma.16G145600). Accordingly, this study exemplified the feasibility of BSR-Seq in studying biotrophic disease resistance in soybean, and showed the genetic makeup of soybean variety 'KS11' comprising the Rmd locus and one MLO gene.

Development and identification of a novel wheat-Thinopyrum ponticum disomic substitution line DS5Ag(5D) with new genes conferring resistance to powdery mildew and leaf rust.

BACKGROUND: Powdery mildew (caused by Blumeria graminis f. sp. tritici (Bgt)) and leaf rust (caused by Puccinia triticina (Pt)) are prevalent diseases in wheat (Triticum aestivum L.) production. Thinopyrum ponticum (2n = 10x = 70, EeEeEbEbExExStStStSt) contains genes that confer high levels of resistance to these diseases. RESULTS: An elite wheat-Th. ponticum disomic substitution line, DS5Ag(5D), was developed in the Bainong Aikang 58 (AK58) background. The line was assessed using genomic in situ hybridization (GISH), oligo-nucleotide probe multiplex (ONPM) fluorescence in situ hybridization (FISH), and molecular markers. Twenty eight chromosome-specific molecular markers were identified for the alien chromosome, and 22 of them were co-dominant. Additionally, SNP markers from the wheat 660 K SNP chip were utilized to confirm chromosome identification and they provide molecular tools for tagging the chromosome in concern. The substitution line demonstrated high levels of resistance to powdery mildew throughout its growth period and to leaf rust at the adult stage. Based on the resistance evaluation of five F5 populations between the substitution lines and wheat genotypes with different levels of sensitivity to the two diseases. Results showed that the resistance genes located on 5Ag confered stable resistance against both diseases across different backgrounds. Resistance spectrum analysis combined with diagnostic marker detection of known resistance genes of Th. ponticum revealed that 5Ag contained two novel genes, Pm5Ag and Lr5Ag, which conferred resistance to powdery mildew and leaf rust, respectively. CONCLUSIONS: In this study, a novel wheat-Th. ponticum disomic substitution line DS5Ag(5D) was successfully developed. The Th. ponticum chromosome 5Ag contain new resistance genes for powdery mildew and leaf rust. Chromosomic-specific molecular markers were generated and they can be used to track the 5Ag chromosome fragments. Consequently, this study provides new elite germplasm resources and molecular markers to facilitate the breeding of wheat varieties that is resistant to powdery mildew and leaf rust.

Genetic mapping of loci affecting seedling and adult-plant resistance to powdery mildew derived from two CIMMYT wheat lines.

MAIN CONCLUSION: PM3 and PM8 alleles carried by two CIMMYT wheat lines confer powdery mildew resistance in seedlings and/or adult plants. A stage-specific epistatic interaction was observed between PM3 and PM8. Powdery mildew is an important foliar disease of wheat. Major genes for resistance, which have been widely used in wheat breeding programs, are typically effective against only limited numbers of virulence genes of the pathogen. The main aim of this study was to map resistance loci in wheat lines 7HRWSN58 and ZWW09-149 from the International Maize and Wheat Improvement Center (CIMMYT). Doubled haploid populations (Magenta/7HRWSN58 and Emu Rock/ZWW09-149) were developed and grown in controlled environment experiments and inoculated with a composite of Blumeria graminis f.sp. tritici isolates that had been collected at various locations in Western Australia. Plants were assessed for powdery mildew symptoms (percentage leaf area diseased) on seedlings and adult plants. Populations were subjected to genotyping-by-sequencing and assayed for known SNPs in the resistance gene PM3. Linkage maps were constructed, and markers were anchored to the wheat reference genome sequence. In both populations, there were asymptomatic lines that exhibited no symptoms. Among symptomatic lines, disease severity varied widely. In the Magenta/7HRWSN58 population, most of the observed variation was attributed to the PM3 region of chromosome 1A, with the allele from 7HRWSN58 conferring resistance in seedlings and adult plants. In the Emu Rock/ZWW09-149 population, two interacting quantitative trait loci were mapped: one at PM3 and the other on chromosome 1B. The Emu Rock/ZWW09-149 population was confirmed to segregate for a 1BL·1RS translocation that carries the PM8 powdery mildew resistance gene from rye. Consistent with previous reports that PM8-derived resistance can be suppressed by PM3 alleles, the observed interaction between the quantitative trait loci on chromosomes 1A and 1B indicated that the PM3 allele carried by ZWW09-149 suppresses PM8-derived resistance from ZWW09-149, but only at the seedling stage. In adult plants, the PM8 region conferred resistance regardless of the PM3 genotype. The resistance sources and molecular markers that were investigated here could be useful in wheat breeding.

Two functional CC-NBS-LRR proteins from rye chromosome 6RS confer differential age-related powdery mildew resistance to wheat.

Rye (Secale cereale), a valuable relative of wheat, contains abundant powdery mildew resistance (Pm) genes. Using physical mapping, transcriptome sequencing, barley stripe mosaic virus-induced gene silencing, ethyl methane sulfonate mutagenesis, and stable transformation, we isolated and validated two coiled-coil, nucleotide-binding site and leucine-rich repeat (CC-NBS-LRR) alleles, PmTR1 and PmTR3, located on rye chromosome 6RS from different triticale lines. PmTR1 confers age-related resistance starting from the three-leaf stage, whereas its allele, PmTR3, confers typical all-stage resistance, which may be associated with their differential gene expression patterns. Overexpression in Nicotiana benthamiana showed that the CC, CC-NBS, and CC-LRR fragments of PMTR1 induce cell death, whereas in PMTR3 the CC and full-length fragments perform this function. Luciferase complementation imaging and pull-down assays revealed distinct interaction activities between the CC and NBS fragments. Our study elucidates two novel rye-derived Pm genes and their derivative germplasm resources and provides novel insights into the mechanism of age-related resistance, which can aid the improvement of resistance against wheat powdery mildew.

Identification of two key genes involved in flavonoid catabolism and their different roles in apple resistance to biotic stresses.

Biosynthesis of flavonoid aglycones and glycosides is well established. However, key genes involved in their catabolism are poorly understood, even though the products of hydrolysis and oxidation play important roles in plant resistance to biotic stress. Here, we report on catabolism of dihydrochalcones (DHCs), the most abundant flavonoids in domesticated apple and wild Malus. Two key genes, BGLU13.1 and PPO05, were identified by activity-directed protein purification. BGLU13.1-A hydrolyzed phlorizin, (the most abundant DHC in domesticated apple) to produce phloretin which was then oxidized by PPO05. The process differed in some wild Malus, where trilobatin (a positional isomer of phlorizin) was mainly oxidized by PPO05. The effects of DHC catabolism on apple resistance to biotic stresses was investigated using transgenic plants. Either directly or indirectly, phlorizin hydrolysis affected resistance to the phytophagous pest two-spotted spider mite, while oxidation of trilobatin was involved in resistance to the biotrophic fungus Podosphaera leucotricha. DHC catabolism did not affect apple resistance to necrotrophic pathogens Valsa mali and Erwinia amylovara. These results suggest that different DHC catabolism pathways play different roles in apple resistance to biotic stresses. The role of DHC catabolism on apple resistance appeared closely related to the mode of invasion/damage used by pathogen/pest.

TaNAC1 boosts powdery mildew resistance by phosphorylation-dependent regulation of TaSec1a and TaCAMTA4 via PP2Ac/CDPK20.

The integrity of wheat (Triticum aestivum) production is increasingly jeopardized by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), particularly amid the vicissitudes of climate change. Here, we delineated the role of a wheat transcription factor, TaNAC1, which precipitates cellular apoptosis and fortifies resistance against Bgt. Utilizing BiFC, co-immunoprecipitation, protein quantification, luciferase report assays, we determined that cytoplasmic TaNAC1-7A undergoes phosphorylation at the S184/S258 sites by TaCDPK20, facilitating its nuclear translocation. This migration appears to prime further phosphorylation by TaMPK1, thereby enhancing transcriptional regulatory activity. Notably, the apoptotic activity of phosphorylated TaNAC1-7A is negatively modulated by the nuclear protein phosphatase PP2Ac. Furthermore, activation of TaNAC1 phosphorylation initiates transcription of downstream genes TaSec1a and TaCAMTA4, through binding to the C[T/G]T[N7]A[A/C]G nucleic acid motif. Suppression of TaNAC1, TaCDPK20, and TaMPK1 in wheat compromises its resistance to Bgt strain E09, whereas overexpression of TaNAC1 and silencing of PP2Ac markedly elevate resistance levels. Our results reveal the pivotal role of TaNAC1 in basal resistance which is mediated by its effects on homotypic fusion, vacuolar protein sorting, and the expression of defense-related genes. The findings highlight the potential through targeting TaNAC1 and its regulators as a strategy for improving wheat's resistance to fungal pathogens.

A fungal plant pathogen overcomes mlo-mediated broad-spectrum disease resistance by rapid gene loss.

Hosts and pathogens typically engage in a coevolutionary arms race. This also applies to phytopathogenic powdery mildew fungi, which can rapidly overcome plant resistance and perform host jumps. Using experimental evolution, we show that the powdery mildew pathogen Blumeria hordei is capable of breaking the agriculturally important broad-spectrum resistance conditioned by barley loss-of-function mlo mutants. Partial mlo virulence of evolved B. hordei isolates is correlated with a distinctive pattern of adaptive mutations, including small-sized (c. 8-40 kb) deletions, of which one is linked to the de novo insertion of a transposable element. Occurrence of the mutations is associated with a transcriptional induction of effector protein-encoding genes that is absent in mlo-avirulent isolates on mlo mutant plants. The detected mutational spectrum comprises the same loci in at least two independently isolated mlo-virulent isolates, indicating convergent multigenic evolution. The mutational events emerged in part early (within the first five asexual generations) during experimental evolution, likely generating a founder population in which incipient mlo virulence was later stabilized by additional events. This work highlights the rapid dynamic genome evolution of an obligate biotrophic plant pathogen with a transposon-enriched genome.

Spatial regulation of immunity: unmasking the secrets of abaxial immunity to powdery mildew.

This article comments on: Wu Y, Sexton WK, Zhang Q, Bloodgood D, Wu Y, Hooks C, Coker F, Vasquez A, Wei C-I, Xiao S. 2024. Leaf abaxial immunity to powdery mildew in Arabidopsis is conferred by multiple defense mechanisms. Journal of Experimental Botany 75, 1465-1478.

Jasmonates, gibberellins, and powdery mildew modify cell cycle progression and evoke differential spatiotemporal responses along the barley leaf.

Barley (Hordeum vulgare) is an important cereal crop, and its development, defence, and stress responses are modulated by different hormones including jasmonates (JAs) and the antagonistic gibberellins (GAs). Barley productivity is severely affected by the foliar biotrophic fungal pathogen Blumeria hordei. In this study, primary leaves were used to examine the molecular processes regulating responses to methyl-jasmonate (MeJA) and GA to B. hordei infection along the leaf axis. Flow cytometry, microscopy, and spatiotemporal expression patterns of genes associated with JA, GA, defence, and the cell cycle provided insights on cell cycle progression and on the gradient of susceptibility to B. hordei observed along the leaf. Notably, the combination of B. hordei with MeJA or GA pre-treatment had a different effect on the expression patterns of the analysed genes compared to individual treatments. MeJA reduced susceptibility to B. hordei in the proximal part of the leaf blade. Overall, distinctive spatiotemporal gene expression patterns correlated with different degrees of cell proliferation, growth capacity, responses to hormones, and B. hordei infection along the leaf. Our results highlight the need to further investigate differential spatial and temporal responses to pathogens at the organ, tissue, and cell levels in order to devise effective disease control strategies in crops.

Pyramiding of transgenic immune receptors from primary and tertiary wheat gene pools improves powdery mildew resistance in the field.

Introgression of resistance genes from wild or related species is a common strategy to improve disease resistance of wheat cultivars. Pm17 is a gene that confers powdery mildew resistance in wheat. It encodes an NLR type of immune receptor and was introgressed from rye to wheat as part of the 1RS chromosome arm translocation several decades ago. So far it has not been possible to separate Pm17 from its co-introgressed rye genes due to suppressed recombination. Here we tested in the field transgenic Bobwhite wheat overexpressing Pm17 without any other rye genes. Four transgenic events showed high levels of PM17 protein accumulation, strong powdery mildew resistance, and no pleiotropic effects during three field seasons. We used a combined approach of transgene insertion and cross-breeding to generate lines co-expressing Pm17 and Pm3, or Pm17 and Pm8. Blumeria graminis f. sp. tritici infection tests confirmed additive, race-specific resistance of the two pyramided transgenes in lines Pm17+Pm3b and Pm17+Pm8. Furthermore, pyramided lines showed strong powdery mildew resistance during three field seasons. We conclude that the combination of overexpressed NLR genes from the extended gene pool broadens and diversifies wheat disease resistance.

Leaf abaxial immunity to powdery mildew in Arabidopsis is conferred by multiple defense mechanisms.

Powdery mildew fungi are obligate biotrophic pathogens that only invade plant epidermal cells. There are two epidermal surfaces in every plant leaf: the adaxial (upper) side and the abaxial (lower) side. While both leaf surfaces can be susceptible to adapted powdery mildew fungi in many plant species, there have been observations of leaf abaxial immunity in some plant species including Arabidopsis. The genetic basis of such leaf abaxial immunity remains unknown. In this study, we tested a series of Arabidopsis mutants defective in one or more known defense pathways with the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. We found that leaf abaxial immunity was significantly compromised in mutants impaired for both the EDS1/PAD4- and PEN2/PEN3-dependent defenses. Consistently, expression of EDS1-yellow fluorescent protein and PEN2-green fluorescent protein fusions from their respective native promoters in the respective eds1-2 and pen2-1 mutant backgrounds was higher in the abaxial epidermal cells than in the adaxial epidermal cells. Altogether, our results indicate that leaf abaxial immunity against powdery mildew in Arabidopsis is at least partially due to enhanced EDS1/PAD4- and PEN2/PEN3-dependent defenses. Such transcriptionally pre-programmed defense mechanisms may underlie leaf abaxial immunity in other plant species such as hemp and may be exploited for engineering adaxial immunity against powdery mildew fungi in crop plants.

Characterization of the powdery mildew resistance locus in wheat breeding line Jimai 809 and its breeding application.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a severe disease that affects the yield and quality of wheat. Popularization of resistant cultivars in production is the preferred strategy to control this disease. In the present study, the Chinese wheat breeding line Jimai 809 showed excellent agronomic performance and high resistance to powdery mildew at the whole growth stage. To dissect the genetic basis for this resistance, Jimai 809 was crossed with the susceptible wheat cultivar Junda 159 to produce segregation populations. Genetic analysis showed that a single dominant gene, temporarily designated PmJM809, conferred the resistance to different Bgt isolates. PmJM809 was then mapped on the chromosome arm 2BL and flanked by the markers CISSR02g-1 and CIT02g-13 with genetic distances 0.4 and 0.8 cM, respectively, corresponding to a physical interval of 704.12-708.24 Mb. PmJM809 differed from the reported Pm genes on chromosome arm 2BL in origin, resistance spectrum, physical position and/or genetic diversity of the mapping interval, also suggesting PmJM809 was located on a complex interval with multiple resistance genes. To analyze and screen the candidate gene(s) of PmJM809, six genes related to disease resistance in the candidate interval were evaluated their expression patterns using an additional set of wheat samples and time-course analysis post-inoculation of the Bgt isolate E09. As a result, four genes were speculated as the key candidate or regulatory genes. Considering its comprehensive agronomic traits and resistance findings, PmJM809 was expected to be a valuable gene resource in wheat disease resistance breeding. To efficiently transfer PmJM809 into different genetic backgrounds, 13 of 19 closely linked markers were confirmed to be suitable for marker-assisted selection. Using these markers, a series of wheat breeding lines with harmonious disease resistance and agronomic performance were selected from the crosses of Jimai 809 and several susceptible cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01467-8.

The mutant STAY-GREEN (Cssgr) in cucumber interacts with the CSEP30 protein to elicit a defense response against Podosphaera xanthii.

Disease-resistant plants activate immune responses by specifically recognition Candidate Secreted Effector Proteins (CSEPs) through resistance (R) proteins. In research on cucumber powdery mildew resistance breeding, several R genes and CSEPs have been identified; however, the specific interactions between R proteins and CSEPs are still largely unexplored. In this study, we used a luciferase reporter assay to identify six CSEPs from Podosphaera xanthii that potentially induce cell death in cucumber. Subsequent yeast two-hybrid analysis revealed that only the mature form of CSEP30 (CSEP30∆SP) interacted with the cucumber mutant STAY-GREEN (Cssgr), a gene previously recognized for its broad-spectrum resistance in genetic studies. This interaction was confirmed using pull-down and co-immunoprecipitation assays. Additionally, to determine if the interaction leads to phenotypic changes, Cssgr and CSEP30∆SP were transiently expressed in tobacco leaves. The infiltration of Cssgr in tobacco resulted in reduced chlorosis compared to the wild-type CsSGR. Co-infiltration of Cssgr with CSEP30∆SP induced distinct dry necrotic lesions, contrasting the effects observed when Cssgr and CSEP30∆SP were infiltrated separately. Additionally, after P. xanthii infection in moderately powdery mildew-resistant Gy14 cucumber, similar necrotic lesions and specific expression of Cssgr, as along with defense response-related genes (CsPR1 and CsLecRK6.1), were observed. This study suggests that the interaction between Cssgr and CSEP30∆SP could trigger cell death and defense response, offering new insights into the molecular function of Cssgr in disease resistance in Gy14 cucumber. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01504-6.

Rapid immunochemical methods for the analysis of proquinazid in strawberry QuEChERS extracts.

Proquinazid is a new-generation fungicide authorized in the EU for combating powdery mildew infections in high-value crops. Due to the perishable nature of fruits, alternative analytical methods are necessary to protect consumer's health from pesticide residues. Currently, immunoassays are a well-established approach for rapidly monitoring chemical contaminants. However, the production of high-quality immunoreagents, such as antibodies and bioconjugates, is essential. This study presents a newly designed hapten that maintains the characteristic moieties of proquinazid unmodified. The linear aliphatic substituents of this molecule were used to introduce the spacer arm. A three-step synthesis strategy was optimized to prepare a hapten that displays the entire 6-iodoquinazolin-4(3H)-one moiety with excellent yields. The N-hydroxysuccimidyl ester of the hapten was activated and purified to prepare a protein conjugate with high hapten density, which was used as an immunogen. Antibodies were raised and competitive enzyme-linked immunosorbent assays were developed. To enhance the assay's sensitivity, two additional heterologous haptens were prepared by modifying the halogenated substituent at C-6. The optimized assays demonstrated low limits of detection in buffer, approximately 0.05 µg/L. When applied to the analysis of proquinazid in QuEChERS extracts of strawberry samples, the immunoassays produced precise and accurate results, particularly in the 10-1000 µg/kg range.