The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes

Plants employ multiple cell‐autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre‐invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane‐localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence‐related vesicle‐resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant–fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle‐mediated trafficking of RPW8.2–yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2–YFP within the EHM exceeds vesicle‐mediated replenishment of RPW8.2–YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre‐invasive defense at the cell periphery and post‐invasive defense at the EHM.     

Characterization of six polymorphic microsatellite loci from Ampelomyces quisqualis,intracellular mycoparasite and biocontrol agent of powdery mildew

Ampelomyces spp. are common intracellular mycoparasites of powdery mildews worldwide and a strain has been commercialized as a biocontrol agent against these plant pathogens. In light of recent genetic analyses revealing high internal transcribed spacer sequence variability among Ampelomyces strains on different host plant mildews, yet no sequence variability within the Malus strain, polymorphic microsatellites were required to permit biocontrol and ecological studies of the complex apple/apple mildew/Ampellomyces tritrophic interaction. For this purpose, described here are the primers to amplify six polymorphic microsatellite loci from Ampelomyces quisqualis isolated from mycelia of the apple powdery mildew fungus, Podosphaera leucotricha.     

Expression of the membrane-associated resistance protein RPW8 enhances basal defense against biotrophic pathogens

The powdery mildew resistance genes RPW8.1 and RPW8.2 from Arabidopsis differ from the other isolated plant resistance (R) genes in their predicted protein domains and their resistance spectrum. The two homologous RPW8 genes encode small proteins featuring a predicted amino-terminal transmembrane anchor domain and a coiled-coil domain and confer resistance to a broad spectrum of powdery mildews. Here, we show that Arabidopsis plants expressing the RPW8 genes have enhanced resistance to another biotrophic pathogen, Hyaloperonospora parasitica, raising the possibility that the RPW8 genes may function to enhance salicylic-acid-dependent basal defenses, rather than as powdery-mildew-specific R genes. When overexpressed from their native promoters, the RPW8 genes confer enhanced resistance to the Cauliflower mosaic virus, but render plants more susceptible to the necrotrophic fungal pathogens Alternaria and Botrytis spp. Furthermore, we show that the RPW8 proteins appear to be localized to the endomembrane system, overlapping with the endoplasmic reticulum-associated small GTPase SAR1, and accumulate to higher levels in response to application of exogenous salicylic acid, one of the signaling molecules of plant defense.     

Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms-0 to two powdery mildew diseases

Arabidopsis thaliana accession La-er was susceptible, and accession Ms-0 was resistant, to powdery mildew diseases caused by Erysiphe cruciferarum UEA1 and E. cichoracearum UCSC1. The resistance reaction phenotype of A. thaliana Ms-0 to both pathogens was characterized, and the resistance loci were genetically mapped. Growth of E. cruciferarum UEA1 on Ms-0 leaves was arrested after formation of the first appressorium: the underlying host epidermal cell collapsed, and occasionally there was necrosis of one or two host mesophyll cells. Growth of E. cichoracearum UCSC1 on Ms-0 leaves was arrested after emergence of several germ tubes from the conidium, and there was necrosis of host mesophyll cells at the sites of infection. Examination of F₂ progeny of a cross La-er x Ms-0 indicated that two independently-segregating dominant loci were required for resistance to E. cruciferarum UEA1. One locus, named RPW6, was genetically mapped to chromosome 5, in a 5.6 cM interval flanked by pCITf16 and PI. The other locus, named RPW7, mapped to chromosome 3 in a 8.5 cM interval flanked by CDC2A and AFC1. Independent effects of RPW6 and RPW7 on E. cruciferarum UEA1 could be detected by quantitative measurements of growth of mycelium and production of conidia. Resistance to E. cichoracearum UCSC1 mapped to a single locus, named RPW8, at a location on chromosome 3 which we could not distinguish from RPW7. Evidently, RPW7 and RPW8 define either a complex resistance locus, or a common resistance gene with dual specificity.     

Interspecific and intraspecific diversity in oak powdery mildews in Europe: coevolution history and adaptation to their hosts

Quercus has been reported as the genus with the largest number of attacking powdery mildews. In Europe, oak powdery mildew was rarely reported before 1907, when severe outbreaks were observed. These epidemics were attributed to the newly described species Erysiphe alphitoides, presumed to be of exotic origin. After the burst of interest following the emergence of the disease, research on this topic remained very limited. Interest in research was recently reactivated in response to the availability of molecular tools. This review summarizes current knowledge on the diversity of oak powdery mildews in Europe and their possible evolutionary relationships with European oaks. The most striking results are the evidence of cryptic diversity (detection in France of a lineage closely related to Erysiphe quercicola, previously thought to only have an Asian distribution), large host range (similarity of E. alphitoides and E. quercicola with powdery mildews of tropical plants) but also local adaptation to Quercus robur. These recent findings highlight the complexity of the history of oak powdery mildew in Europe and point to the question of host specialization and host jumps in the evolution of powdery mildew fungi.     

A Comprehensive Mutational Analysis of the Arabidopsis Resistance Protein RPW8.2 Reveals Key Amino Acids for Defense Activation and Protein Targeting

The Arabidopsis thaliana RESISTANCE TO POWDERY MILDEW8.2 (RPW8.2) protein is specifically targeted to the extrahaustorial membrane (EHM) encasing the haustorium, or fungal feeding structure, where RPW8.2 activates broad-spectrum resistance against powdery mildew pathogens. How RPW8.2 activates defenses at a precise subcellular locale is not known. Here, we report a comprehensive mutational analysis in which more than 100 RPW8.2 mutants were functionally evaluated for their defense and trafficking properties. We show that three amino acid residues (i.e., threonine-64, valine-68, and aspartic acid-116) are critical for RPW8.2-mediated cell death and resistance to powdery mildew (Golovinomyces cichoracearum UCSC1). Also, we reveal that two arginine (R)– or lysine (K)–enriched short motifs (i.e., R/K-R/K-x-R/K) make up the likely core EHM-targeting signals, which, together with the N-terminal transmembrane domain, define a minimal sequence of 60 amino acids that is necessary and sufficient for EHM localization. In addition, some RPW8.2 mutants localize to the nucleus and/or to a potentially novel membrane that wraps around plastids or plastid-derived stromules. Results from this study not only reveal critical amino acid elements in RPW8.2 that enable haustorium-targeted trafficking and defense, but also provide evidence for the existence of a specific, EHM-oriented membrane trafficking pathway in leaf epidermal cells invaded by powdery mildew.     

Strong population genetic structure of an invasive species,Rhynchophorus ferrugineus (Olivier), in southern China

The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier), was initially reported in China in the 1990s and is now considered one of the most successful invasive pests of palm plants in the country. A total of 14 microsatellite loci and one mitochondrial cytochrome oxidase subunit Ι (cox I) gene fragment were used to investigate the genetic characteristics and structure of R. ferrugineus in southern China. High levels of genetic differentiation among populations and significant correlations between genetic and geographical distances indicated an important role of geographical distance in the distribution of the RPW in southern China. High gene flow between Fujian and Taiwan province populations illustrated the increased effects of frequent anthropogenic activities on gene flow between them. Genetic similarity (i.e., haplotype similarity) indicated that RPW individuals from Taiwan and Fujian invaded from a different source than those from Hainan. To some extent, the genetic structure of the RPW in southern China correlated well with the geographic origins of this pest. We propose that geographical distance, anthropogenic activities, and the biological attributes of this pest are responsible for the distribution pattern of the RPW in southern China. The phylogenetic analysis suggests that the most likely native sources of the RPW in southern China are India, the Philippines, and Vietnam.     

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.

     

Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens

In previous work, UEA1 and UCSC1, two geographically distinct, powdery mildew isolates, were recognized for their ability to infect Arabidopsis thaliana. We have clarified the identity of these isolates by determining their host ranges, reexamining their morphology, and comparing their DNA sequences for the 5.8S ribosomal RNA and two flanking internal transcribed spacer sequences. These experiments confirm that UEA1 is a member of Erysiphe cruciferarum and that UCSC1 belongs to E. cichoracearum. Interactions of the two Erysiphe isolates with 360 A. thaliana accessions were examined to provide a comprehensive profile of naturally occurring powdery mildew resistance in this weedy species. The majority of A. thaliana accessions (213) were susceptible to both isolates. Among the accessions exhibiting some degree of resistance, most (84) responded differentially to UEA1 and UCSC1 and the remainder were resistant to both isolates. Notably, resistance to UCSC1 cosegregated with RPW7, a locus previously demonstrated to confer resistance to UEA1 in Ms-0 x Landsberg (erecta) crosses. With this large collection of resistant accessions, questions about species specificity, genetic diversity and the evolution of resistance to powdery mildews can be addressed.     

Origin and maintenance of a broad-spectrum disease resistance locus in Arabidopsis

The broad-spectrum mildew resistance genes RPW8.1 and RPW8.2 define a unique type of plant disease resistance (R) gene, and so far homologous sequences have been found in Arabidopsis thaliana only, which suggests a recent origin. In addition to RPW8.1 and RPW8.2, the RPW8 locus contains three homologs of RPW8, HR1, HR2, and HR3, which do not contribute to powdery mildew resistance. To investigate whether RPW8 has originated recently, and if so the processes involved, we have isolated and analyzed the syntenic RPW8 loci from Arabidopsis lyrata, and from Brassica rapa and B. oleracea. The A. lyrata locus contains four genes orthologous to HR1, HR2, HR3, and RPW8.2, respectively. Two syntenic loci have been characterized in Brassica; one locus contains three genes and is present in both B. oleracea and B. rapa, and the other locus contains a single gene and is detected in B. rapa only. The Brassica homologs have highest similarity to HR3. Sequence analyses suggested that the RPW8 gene family in Brassicaceae originated from an HR3-like ancestor gene through a series of duplications and that RPW8.1 and RPW8.2 evolved from functional diversification through positive selection several MYA. Examination of the sequence polymorphism of 32 A. thaliana accessions at the RPW8 locus and their disease reaction phenotypes revealed that the polymorphic RPW8 locus defines a major source of resistance to powdery mildew diseases. A possible evolutionary mechanism by which functional polymorphism at the AtRPW8 locus has been maintained in contemporary populations of A. thaliana is discussed.     

High microsatellite genetic diversity fails to predict greater population resistance to extreme drought

There is intense debate whether genetic diversity measured via neutral molecular markers can be used as a surrogate for fitness and as an indirect estimate of the amount of genetic variation for fitness-related traits in a population. Here, we measured microsatellite DNA genetic diversity (before the onset of drought) and mortality after prolonged drought in 15 populations of Banksia hookeriana in the species-rich southwestern Australian flora, to test the relationship between population genetic diversity and resistance to extreme climate fluctuations. Number of alleles per locus varied from 5.2 to 8.2 at eleven microsatellite loci among 30 individuals in each population. Mortality varied from 25 to 50% in individual populations after prolonged drought. Lower mortality was not observed in populations with higher genetic diversity, but in populations with lower genetic diversity. Thus, higher microsatellite genetic diversity fails to predict lower population mortality during extreme drought in B. hookeriana. Our results imply that it may be misleading to use studies of neutral genetic variation exclusively as the basis for inferring population and species capacity for resisting extreme climate events and for species conservation and management decisions.     

Evolution of resistance against powdery mildew in winter wheat populations conducted under dynamic management. I – Is specific seedling resistance selected?

Dynamic management has been proposed as a complementary strategy to gene banks for the conservation of genetic resources. The evolution of frequencies of genes for specific resistance towards powdery mildew (caused by Blumeria graminis f. sp. tritici) in populations of a French network for dynamic management of bread wheat genetic resources was investigated after 10 years of multiplication without human selection. The objective was to determine whether specific resistance gene diversity was maintained in the populations and whether any changes could be attributed to selection due to pathogen pressure. Seven populations, originating from four of the network sites, were characterized and compared to the initial population for six specific resistance gene frequencies detected by nine Blumeria graminis f. sp. tritici isolates. Diversity decreased at the population level, but because of a strong differentiation between the populations, this diversity was maintained at the network level. The comparison of Fst parameters estimated on neutral markers (RFLP) and on resistance gene data revealed that in two of the populations specific resistance genes had been selected by pathogen pressure, whereas evolution in two other populations seemed to be the result of genetic drift. For the three last populations, conclusions were less clear, as one had probably experienced a strong bottleneck and the other two presented intermediate Fst values. A dynamic management network with sites contrasted for pathogen pressure, allowing genetic drift in some populations and selection in others, appeared, at least on the short term, to be a good tool for maintaining the diversity of genes for specific resistance to powdery mildew. Received: 15 December 1999 / Accepted: 30 December 1999     

Natural genetic resources of Arabidopsis thaliana reveal a high prevalence and unexpected phenotypic plasticity of RPW8-mediated powdery mildew resistance

Here, an approach based on natural genetic variation was adopted to analyse powdery mildew resistance in Arabidopsis thaliana. Accessions resistant to multiple powdery mildew species were crossed with the susceptible Col-0 ecotype and inheritance of resistance was analysed. Histochemical staining was used to visualize archetypal plant defence responses such as callose deposition, hydrogen peroxide accumulation and host cell death in a subset of these ecotypes. In six accessions, resistance was likely of polygenic origin while 10 accessions exhibited evidence for a single recessively or semi-dominantly inherited resistance locus. Resistance in the latter accessions was mainly manifested at the terminal stage of the fungal life cycle by a failure of abundant conidiophore production. The resistance locus of several of these ecotypes was mapped to a genomic region containing the previously analysed atypical RPW8 powdery mildew resistance genes. Gene silencing revealed that members of the RPW8 locus were responsible for resistance to Golovinomyces orontii in seven accessions. These results suggest that broad-spectrum powdery mildew resistance in A. thaliana is predominantly of polygenic origin or based on RPW8 function. The findings shed new light on the natural variation of inheritance, phenotypic expression and pathogen range of RPW8-conditioned powdery mildew resistance.     

Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance

The RPW8 locus from Arabidopsis thaliana Ms-0 includes two functional paralogous genes ( RPW8.1 and RPW8.2 ) and confers broad-spectrum resistance via the salicylic acid-dependent signaling pathway to the biotrophic fungal pathogens Golovinomyces spp. that cause powdery mildew diseases on multiple plant species. To identify proteins involved in regulation of the RPW8 protein function, a yeast two-hybrid screen was performed using RPW8.2 as bait. The 14-3-3 isoform lambda (designated GF14λ) was identified as a potential RPW8.2 interactor. The RPW8.2–GF14λ interaction was specific and engaged the C-terminal domain of RPW8.2, which was confirmed by pulldown assays. The physiological impact of the interaction was revealed by knocking down GF14λ by T-DNA insertion, which compromised basal and RPW8-mediated resistance to powdery mildew. In addition, over-expression of GF14λ resulted in hypersensitive response-like cell death and enhanced resistance to powdery mildew via the salicylic acid-dependent signaling pathway. The results from this study suggest that GF14λ may positively regulate the RPW8.2 resistance function and play a role in enhancing basal resistance in Arabidopsis.     

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.     

Length polymorphism and allele structure of trinucleotide microsatellites in natural accessions of Arabidopsis thaliana

 The objective of this work was to assess the degree of trinucleotide microsatellite length polymorphism in the selfing species Arabidopsis thaliana. PCR amplifications of 12 microsatellite loci among 49 natural populations revealed between one to eight length variants (alleles) for each locus. The average number of alleles per locus was four and the average genetic diversity index was 0.43. Divergence between length variants was investigated at the nucleotide level. Several observations emerge from the sequence data: (1) for most loci, length polymorphism results only from variations in the number of trinucleotide repeats; (2) for a few others, some variability was noted in the flanking sequences; (3) for compound and interrupted loci containing two arrays of trinucleotide repeats, length variations preferentially affect the longest one. Five of the Arabidopsis thaliana accessions were clearly composed of two sublines. In 2 other accessions, some heterozygous individual plants, probably resulting from recent outcrosses, were found. A phylogenetic tree constructed on the basis of trinucleotide microsatellite allelic diversity shows that genetic relationships among the accessions are not correlated with their geographic origin. Received: 4 November 1997 / Accepted: 3 March 1998