Thierry C. Marcel

Nonhost and basal resistance: how to explain specificity?

Nonhost resistance to plant pathogens can be constitutive or induced by microbes. Successful pathogens suppress microbe-induced plant defences by delivering appropriate effectors, which are apparently not sufficiently effective on nonhost plant species, as can be concluded from the strong host specificity of many biotroph plant pathogens. Such effectors act on particular plant targets, such as promoters or motifs in expressed sequences. Despite much progress in the elucidation of the molecular aspects of nonhost resistance to plant pathogens, very little is known about the genes that determine whether effectors can or cannot suppress the basal defence. In hosts they can, in nonhosts they cannot. The targets determining the host status of plants can be identified in inheritance studies. Recent reports have indicated that nonhost resistance is inherited polygenically, and exhibits strong similarity and association with the basal resistance of plants to adapted pathogens.

Tomato Defense to Oidium neolycopersici: Dominant Ol Genes Confer Isolate-Dependent Resistance Via a Different Mechanism Than Recessive ol-2

Tomato powdery mildew caused by Oidium neolycopersici has become a globally important disease of tomato (Lycopersicon esculentum). To study the defense responses of tomato triggered by tomato powdery mildew, we first mapped a set of resistance genes to O. neolycopersici from related Lycopersicon species. An integrated genetic map was generated showing that all the dominant resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) are located on tomato chromosome 6 and are organized in three genetic loci. Then, near-isogenic lines (NIL) were produced that contain the different dominant Ol genes in a L. esculentum genetic background. These NIL were used in disease tests with local isolates of O. neolycopersici in different geographic locations, demonstrating that the resistance conferred by different Ol genes was isolate-dependent and, hence, may be race-specific. In addition, the resistance mechanism was analyzed histologically. The mechanism of resistance conferred by the dominant Ol genes was associated with hypersensitive response, which varies in details depending on the Ol-gene in the NIL, while the mechanism of resistance governed by the recessive gene ol-2 on tomato chromosome 4 was associated with papillae formation.

Basal Host Resistance of Barley to Powdery Mildew: Connecting Quantitative Trait Loci and Candidate Genes

The basal resistance of barley to powdery mildew (Blumeria graminis f. sp. hordei) is a quantitatively inherited trait that is based on nonhypersensitive mechanisms of defense. A functional genomic approach indicates that many plant candidate genes are involved in the defense against formation of fungal haustoria. It is not known which of these candidate genes have allelic variation that contributes to the natural variation in powdery mildew resistance, because many of them may be highly conserved within the barley species and may act downstream of the basal resistance reaction. Twenty-two expressed sequence tag or cDNA clone sequences that are likely to play a role in the barley-Blumeria interaction based on transcriptional profiling, gene silencing, or overexpression data, as well as mlo, Ror1, and Ror2, were mapped and considered candidate genes for contribution to basal resistance. We mapped the quantitative trait loci (QTL) for powdery mildew resistance in six mapping populations of barley at seedling and adult plant stages and developed an improved high-density integrated genetic map containing 6,990 markers for comparing QTL and candidate gene positions over mapping populations. We mapped 12 QTL at seedling stage and 13 QTL at adult plant stage, of which four were in common between the two developmental stages. Six of the candidate genes showed coincidence in their map positions with the QTL identified for basal resistance to powdery mildew. This co-localization justifies giving priority to those six candidate genes to validate them as being responsible for the phenotypic effects of the QTL for basal resistance.

High diversity of genes for nonhost resistance of barley to heterologous rust fungi.

Inheritance studies on the nonhost resistance of plants would normally require interspecific crosses that suffer from sterility and abnormal segregation. Therefore, we developed the barley–Puccinia rust model system to study, using forward genetics, the specificity, number, and diversity of genes involved in nonhost resistance. We developed two mapping populations by crossing the line SusPtrit, with exceptional susceptibility to heterologous rust species, with the immune barley cultivars Vada and Cebada Capa. These two mapping populations along with the Oregon Wolfe Barley population, which showed unexpected segregation for resistance to heterologous rusts, were phenotyped with four heterologous rust fungal species. Positions of QTL conferring nonhost resistance in the three mapping populations were compared using an integrated consensus map. The results confirmed that nonhost resistance in barley to heterologous rust species is controlled by QTL with different and overlapping specificities and by an occasional contribution of an R-gene for hypersensitivity. In each population, different sets of loci were implicated in resistance. Few genes were common between the populations, suggesting a high diversity of genes conferring nonhost resistance to heterologous pathogens. These loci were significantly associated with QTL for partial resistance to the pathogen Puccinia hordei and with defense-related genes.

An eQTL Analysis of Partial Resistance to Puccinia hordei in Barley

Background Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes. Methodology/Principal Findings We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis. Conclusions/Significance The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this population.

Peroxidase profiling reveals genetic linkage between peroxidase gene clusters and basal host and non-host resistance to rusts and mildew in barley.

Background Higher plants possess a large multigene family encoding secreted class III peroxidase (Prx) proteins. Peroxidases appear to be associated with plant disease resistance based on observations of induction during disease challenge and the presence or absence of isozymes in resistant vs susceptible varieties. Despite these associations, there is no evidence that allelic variation of peroxidases directly determines levels of disease resistance. Methodology/Principal Findings The current study introduces a new strategy called Prx-Profiling. We showed that with this strategy a large number of peroxidase genes can be mapped on the barley genome. In order to obtain an estimate of the total number of Prx clusters we followed a re-sampling procedure, which indicated that the barley genome contains about 40 peroxidase gene clusters. We examined the association between the Prxs mapped and the QTLs for resistance of barley to homologous and heterologous rusts, and to the barley powdery mildew fungus. We report that 61% of the QTLs for partial resistance to P. hordei, 61% of the QTLs for resistance to B. graminis and 47% of the QTLs for non-host resistance to other Puccinia species co-localize with Prx based markers. Conclusions/Significance We conclude that Prx-Profiling was effective in finding the genetic location of Prx genes on the barley genome. The finding that QTLs for basal resistance to rusts and powdery mildew fungi tend to co-locate with Prx clusters provides a base for exploring the functional role of Prx-related genes in determining natural differences in levels of basal resistance.

Host Status of False Brome Grass to the Leaf Rust Fungus Puccinia brachypodii and the Stripe Rust Fungus P. striiformis

Purple false brome grass (Brachypodium distachyon) has recently emerged as a model system for temperate grasses and is also a potential model plant to investigate plant interactions with economically important pathogens such as rust fungi. We determined the host status of five Brachypodium species to three isolates of Puccinia brachypodii, the prevalent rust species on Brachypodium sylvaticum in nature, and to one isolate each of three formae speciales of the stripe rust fungus P. striiformis. Two P. striiformis isolates produced sporulating lesions, both in only one of the tested interactions, suggesting a marginal host status of B. distachyon. P. brachypodii formed sporulating uredinia on the five Brachypodium species tested, and a range of reactions was observed. Surprisingly, the B. sylvaticum-derived rust isolates were more frequently pathogenic to B. distachyon than to their original host species. The B. distachyon diploid inbred lines, developed and distributed as reference material to the Brachypodium research community, include susceptible and resistant genotypes to at least three of the four P. brachypodii isolates tested. This creates the opportunity to use B. distachyon/P. brachypodii as a model pathosystem. In one B. distachyon accession, heavy infection by the loose smut fungus Ustilago bromivora occurred. That pathogen could also serve as a model pathogen of Brachypodium.

Evidence for a Minor Gene-for-Minor Gene Interaction Explaining Nonhypersensitive Polygenic Partial Disease Resistance

ABSTRACT Partial resistance is a quantitative type of resistance that, by definition of Parlevliet, is not based on hypersensitivity. It is largely pathotype nonspecific, although some minor isolate-specific responses have been reported. In order to elucidate the isolate specificity of individual genes for partial resistance, three barley recombinant inbred line mapping populations were analyzed for resistance to the leaf rust fungus Puccinia hordei. The mapping populations were inoculated with one isolate avirulent and two isolates virulent to resistance gene Rph7g. Six significant quantitative trait loci (QTLs) were detected. Of these, two (Rphq3 and Rphq11) were detected with only the avirulent isolate (1.2.1.) and one (Rphq18) only with both virulent isolates (CO-04 and 28.1). The effectiveness of these QTLs was tested with 14 isolates, using a tester set of genotypes containing alleles for resistance or susceptibility for these QTLs. QTL Rphq18 was effective to only two isolates, CO-04 and 28.1, whereas Rphq3 and Rphq11 were ineffective to CO-04 and 28.1 but effective to all other isolates, except one. This resulted in a significant Persons differential interaction, which is a hallmark of a gene-for-gene interaction. The minor gene-for-minor gene interaction is not based on hypersensitivity and there is no evidence that the resistance is based on genes belonging to the nucleotide-binding leucine-rich repeat class.

The phenotypic expression of QTLs for partial resistance to barley leaf rust during plant development

Partial resistance is generally considered to be a durable form of resistance. In barley, Rphq2, Rphq3 and Rphq4 have been identified as consistent quantitative trait loci (QTLs) for partial resistance to the barley leaf rust pathogen Puccinia hordei. These QTLs have been incorporated separately into the susceptible L94 and the partially resistant Vada barley genetic backgrounds to obtain two sets of near isogenic lines (NILs). Previous studies have shown that these QTLs are not effective at conferring disease resistance in all stages of plant development. In the present study, the two sets of QTL–NILs and the two recurrent parents, L94 and Vada, were evaluated for resistance to P. hordei isolate 1.2.1 simultaneously under greenhouse conditions from the first leaf to the flag leaf stage. Effect of the QTLs on resistance was measured by development rate of the pathogen, expressed as latency period (LP). The data show that Rphq2 prolongs LP at the seedling stage (the first and second leaf stages) but has almost no effect on disease resistance in adult plants. Rphq4 showed no effect on LP until the third leaf stage, whereas Rphq3 is consistently effective at prolonging LP from the first leaf to the flag leaf. The changes in the effectiveness of Rphq2 and Rphq4 happen at the barley tillering stage (the third to fourth leaf stages). These results indicate that multiple disease evaluations of a single plant by repeated inoculations of the fourth leaf to the flag leaf should be conducted to precisely estimate the effect of Rphq4. The present study confirms and describes in detail the plant development-dependent effectiveness of partial resistance genes and, consequently, will enable a more precise evaluation of partial resistance regulation during barley development.

Differential gene expression in nearly isogenic lines with QTL for partial resistance to Puccinia hordei in barley

BackgroundThe barley-Puccinia hordei (barley leaf rust) pathosystem is a model for investigating partial disease resistance in crop plants and genetic mapping of phenotypic resistance has identified several quantitative trait loci (QTL) for partial resistance. Reciprocal QTL-specific near-isogenic lines (QTL-NILs) have been developed that combine two QTL, Rphq 2 and Rphq 3, the largest effects detected in a recombinant-inbred-line (RIL) population derived from a cross between the super-susceptible line L94 and partially-resistant line Vada. The molecular mechanism underpinning partial resistance in these QTL-NILs is unknown.ResultsAn Agilent custom microarray consisting of 15,000 probes derived from barley consensus EST sequences was used to investigate genome-wide and QTL-specific differential expression of genes 18 hours post-inoculation (hpi) with Puccinia hordei. A total of 1,410 genes were identified as being significantly differentially expressed across the genome, of which 55 were accounted for by the genetic differences defined by QTL-NILs at Rphq 2 and Rphq 3. These genes were predominantly located at the QTL regions and are, therefore, positional candidates. One gene, encoding the transcriptional repressor Ethylene-Responsive Element Binding Factor 4 (HvERF4) was located outside the QTL at 71 cM on chromosome 1H, within a previously detected eQTL hotspot for defence response. The results indicate that Rphq 2 or Rphq 3 contains a trans-eQTL that modulates expression of HvERF4. We speculate that HvERF4 functions as an intermediate that conveys the response signal from a gene(s) contained within Rphq 2 or Rphq 3 to a host of down-stream defense responsive genes. Our results also reveal that barley lines with extreme or intermediate partial resistance phenotypes exhibit a profound similarity in their spectrum of Ph-responsive genes and that hormone-related signalling pathways are actively involved in response to Puccinia hordei.ConclusionsDifferential gene expression between QTL-NILs identifies genes predominantly located within the target region(s) providing both transcriptional and positional candidate genes for the QTL. Genetically mapping the differentially expressed genes relative to the QTL has the potential to discover trans-eQTL mediated regulatory relays initiated from genes within the QTL regions.