ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt

Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.     

HOW SPECIFICITY AND EPIDEMIOLOGY DRIVE THE COEVOLUTION OF STATIC TRAIT DIVERSITY IN HOSTS AND PARASITES

There is typically considerable variation in the level of infectivity of parasites and the degree of resistance of hosts within populations. This trait variation is critical not only to the evolutionary dynamics but also to the epidemiology, and potentially the control of infectious disease. However, we lack an understanding of the processes that generate and maintain this trait diversity. We examine theoretically how epidemiological feedbacks and the characteristics of the interaction between host types and parasites strains determine the coevolution of host–parasite diversity. The interactions include continuous characterizations of the key phenotypic features of classic gene‐for‐gene and matching allele models. We show that when there are costs to resistance in the hosts and infectivity in the parasite, epidemiological feedbacks may generate diversity but this is limited to dimorphism, often of extreme types, in a broad range of realistic infection scenarios. For trait polymorphism, there needs to be both specificity of infection between host types and parasite strains as well as incompatibility between particular strains and types. We emphasize that although the high specificity is well known to promote temporal “Red Queen” diversity, it is costs and combinations of hosts and parasites that cannot infect that will promote static trait diversity.     

Within‐ and among‐population variation in infectivity,latency and spore production in a host–pathogen system

In spatially structured populations, host–parasite coevolutionary potential depends on the distribution of genetic variation within and among populations. Inoculation experiments using the plant, Silene latifolia, and its fungal pathogen, Microbotryum violaceum, revealed little overall differentiation in infectivity/resistance, latency or spore production among host or pathogen populations. Within populations, fungal strains had similar means, but varied in performance across plant populations. Variation in resistance among seed families indicates the potential for parasite‐mediated selection, whereas there was little evidence for local pathogen genotype × plant genotype interactions assumed by most theoretical coevolution models. Lower spore production on sympatric than allopatric hosts confirmed local fungal maladaptation already observed for infectivity. Correlations between infectivity and latency or spore production suggest a common mechanism for variation in these traits. Our results suggest low variation available to this pathogen for tracking its coevolving host. This may be caused by random drift, breeding system or migration characteristic of metapopulation dynamics.     

Polygenic traits and parasite local adaptation

The extent to which parasites are locally adapted to their hosts has important implications for human health and agriculture. A recently developed conceptual framework--the geographic mosaic theory of coevolution--predicts that local maladaptation should be common and largely determined by the interplay between gene flow and spatially variable reciprocal selection. Previous investigation of this theory has predominately focused on genetic systems of infection and resistance characterized by few genes of major effect and particular forms of epistasis. Here we extend existing theory by analyzing mathematical models of host-parasite interactions in which host resistance to parasites is mediated by quantitative traits with an additive polygenic basis. In contrast to previous theoretical studies predicated upon major gene mechanisms, we find that parasite local maladaptation is quite uncommon and restricted to one specific functional form of host resistance. Furthermore, our results show that local maladaptation should be rare or absent in studies that measure local adaptation using reciprocal transplant designs conducted in natural environments. Our results thus narrow the scope over which the predictions of the geographic mosaic theory are likely to hold and provide novel and readily testable predictions about when and where local maladaptation is expected.     

The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana

We have used the naturally occurring plant-parasite system of Arabidopsis thaliana and its common parasite Peronospora parasitica (downy mildew) to study the evolution of resistance specificity in the host population. DNA sequence of the resistance gene, RPP13, from 24 accessions, including 20 from the United Kingdom, revealed amino acid sequence diversity higher than that of any protein coding gene reported so far in A. thaliana. A significant excess of amino acid polymorphism segregating within this species is localized within the leucine-rich repeat (LRR) domain of RPP13. These results indicate that single alleles of the gene have not swept through the population, but instead, a diverse collection of alleles have been maintained. Transgenic complementation experiments demonstrate functional differences among alleles in their resistance to various pathogen isolates, suggesting that the extreme amino acid polymorphism in RPP13 is maintained through continual reciprocal selection between host and pathogen.     

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.     

Resistance of Glycine species and various cultivated legumes to the soybean aphid (Homoptera: Aphididae)

The soybean aphid, Aphis glycines Matsumura, is a new pest of soybean, Glycine max (L.) Merr., in North America. It has become widespread on soybean in North America since it was first identified in the Midwest in 2000. Species of Rhamnus L. (buckthorn) are the primary hosts of A. glycines, and soybean is known as a secondary host. There is limited information about the secondary host range of A. glycines. Aphid colonization on various legume hosts was compared in choice experiments. Aphid colonization occurred on species in the genus Glycine Wild. No colonization occurred on Lablab purpureus (L.) Sweet, Lens culinaris Medik, Phaseolus vulgaris L., Pisum sativum L., or species of Vicia L. and Vigna Savi. Colonization was limited or aphids were transient on species of Medicago L., Phaseolus L., and Trifolium L. There were significant differences in aphid colonization among Medicago truncatula accessions with numbers ranging from 7 to 97 aphids per plant. Six Glycine soja Sieb. & Zucc. accessions were as resistant as G. max accessions to A. glycines; these may represent novel sources of A. glycines resistance not found in G. max. Antibiosis was found to play a large role in the expression of resistance in three of the G. soja accessions. Results of this study indicated that G. max and G. soja were the best secondary hosts of A. glycines; however, its secondary host range may include other leguminous species. Therefore, A. glycines did not seem to have a highly restricted monophagous secondary host range.     

WRR4 encodes a TIR-NB-LRR protein that confers broad-spectrum white rust resistance in Arabidopsis thaliana to four physiological races of Albugo candida

White blister rust in the Brassicaceae is emerging as a superb model for exploring how plant biodiversity has channeled speciation of biotrophic parasites. The causal agents of white rust across a wide breadth of cruciferous hosts currently are named as variants of a single oomycete species, Albugo candida. The most notable examples include a major group of physiological races that each are economically destructive in a different vegetable or oilseed crop of Brassica juncea (A. candida race 2), B. rapa (race 7), or B. oleracea (race 9); or parasitic on wild crucifers such as Capsella bursa-pastoris (race 4). Arabidopsis thaliana is innately immune to these races of A. candida under natural conditions; however, it commonly hosts its own molecularly distinct subspecies of A. candida (A. candida subsp. arabidopsis). In the laboratory, we have identified several accessions of Arabidopsis thaliana (e.g.,. Ws-3) that can permit varying degrees of rust development following inoculation with A. candida races 2, 4, and 7, whereas race 9 is universally incompatible in Arabidopsis thaliana and nonrusting resistance is the most prevalent outcome of interactions with the other races. Subtle variation in resistance phenotypes is evident, observed initially with an isolate of A. candida race 4, indicating additional genetic variation. Therefore, we used the race 4 isolate for map-based cloning of the first of many expected white rust resistance (WRR) genes. This gene was designated WRR4 and encodes a cytoplasmic toll-interleukin receptor-like nucleotide-binding leucine-rich repeat receptor-like protein that confers a dominant, broad-spectrum white rust resistance in the Arabidopsis thaliana accession Columbia to representative isolates of A. candida races 2, 4, 7, and 9, as verified by transgenic expression of the Columbia allele in Ws-3. The WRR4 protein requires functional expression of the lipase-like protein EDS1 but not the paralogous protein PAD4, and confers full immunity that masks an underlying nonhypersensitive incompatibility in Columbia to A. candida race 4. This residual incompatibility is independent of functional EDS1.     

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.     

Medicago truncatula in Interaction with Fusarium and Rhizoctonia Phytopathogenic Fungi: Fungal Aggressiveness,Plant Response Biodiversity and Character Heritability Indices

Fusarium and Rhizoctonia genera are important pathogens of many field crops worldwide. They are constantly evolving and expanding their host range. Selecting resistant cultivars is an effective strategy to break their infection cycles. To this end, we screened a collection of Medicago truncatula accessions against Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani strains isolated from different plant species. Despite the small collection, a biodiversity in the disease response of M. truncatula accessions ranging from resistant phenotypes to highly susceptible ones was observed. A17 showed relative resistance to all fungal strains with the lowest disease incidence and ratings while TN1.11 was among the susceptible accessions. As an initiation of the characterization of resistance mechanisms, the antioxidant enzymes’ activities, at the early stages of infections, were compared between these contrasting accessions. Our results showed an increment of the antioxidant activities within A17 plants in leaves and roots. We also analyzed the responses of a population of recombinant inbred lines derived from the crossing of A17 and TN1.11 to the infection with the same fungal strains. The broad-sense heritability of measured traits ranged from 0.87 to 0.95, from 0.72 to 0.96, and from 0.14 to 0.85 under control, F. oxysporum, and R. solani conditions, respectively. This high estimated heritability underlines the importance of further molecular analysis of the observed resistance to identify selection markers that could be incorporated into a breeding program and thus improving soil-borne pathogens resistance in crops.     

LOCAL MALADAPTATION IN THE ANTHER-SMUT FUNGUS MICROBOTRYUM VIOLACEUM TO ITS HOST PLANT SILENE LATIFOLIA: EVIDENCE FROM A CROSS-INOCULATION EXPERIMENT

Conventional wisdom holds that parasites evolve more rapidly than their hosts and are therefore locally adapted, that is, better at exploiting sympatric than allopatric hosts. We studied local adaptation in the insect-transmitted fungal pathogen Microbotryum violaceum and its host plant Silene latifolia. Infection success was tested in sympatric (local) and allopatric (foreign) combinations of pathogen and host from 14 natural populations from a metapopulation. Seedlings from up to 10 seed families from each population were exposed to sporidial suspensions from each of four fungal strains derived from the same population, from a near-by population (< 10 km distance), and from two populations at an intermediate (< 30 km) and remote (< 170 km) distance, respectively. We obtained significant pathogen X plant interactions in infection success (proportion of diseased plants) at both fungal population and strain level. There was an overall pattern of local maladaptation of this pathogen: average fungal infection success was significantly lower on sympatric hosts (mean proportion of diseased plants = 0.32 ± 0.03 SE) than on allopatric hosts (0.40 ± 0.02). Five of the 14 fungal populations showed no strong reduction in infection success on sympatric hosts, and three even tended to perform better on sympatric hosts. This pattern is consistent with models of time-lagged cycles predicting patterns of local adaptation in host-parasite systems to emerge only on average. Several factors may restrict the evolutionary potential of this pathogen relative to that of its host. First, a predominantly selfing breeding system may limit its ability to generate new virulence types by sexual recombination, whereas the obligately outcrossing host 5. latifolia may profit from rearrangement of resistance alleles by random mating. Second, populations often harbor only a few infected individuals, so virulence variation may be further reduced by drift. Third, migration rates among host plant populations are much higher than among pathogen populations, possibly because pollinators prefer healthy over diseased plants. Migration among partly isolated populations may therefore introduce novel host plant resistance variants more often than novel parasite virulence variants. That migration contributes to the coevolutionary dynamics in this system is supported by the geographic pattern of infectivity. Infection success increased over the first 10–km range of host-pathogen population distances, which is likely the natural range of gene exchange.     

Maintenance of genetic variation in plants and pathogens involves complex networks of gene-for-gene interactions

The RPP13 [recognition of Hyaloperonospora arabidopsidis (previously known as Peronospora parasitica )] resistance ( R ) gene in Arabidopsis thaliana exhibits the highest reported level of sequence diversity among known R genes. Consistent with a co-evolutionary model, the matching effector protein ATR13 ( A. thaliana -recognized) from H. arabidopsidis reveals extreme levels of allelic diversity. We isolated 23 new RPP13 sequences from a UK metapopulation, giving a total of 47 when combined with previous studies. We used these in functional studies of the A. thaliana accessions for their resistance response to 16 isolates of H. arabidopsidis . We characterized the molecular basis of recognition by the expression of the corresponding ATR13 genes from these 16 isolates in these host accessions. This allowed the determination of which alleles of RPP13 were responsible for pathogen recognition and whether recognition was dependent on the RPP13/ATR13 combination. Linking our functional studies with phylogenetic analysis, we determined that: (i) the recognition of ATR13 is mediated by alleles in just a single RPP13 clade; (ii) RPP13 alleles in other clades have evolved the ability to detect other pathogen ATR protein(s); and (iii) at least one gene, unlinked to RPP13 in A. thaliana, detects a different subgroup of ATR13 alleles.     

Natural variation in Arabidopsis thaliana as a tool for highlighting differential drought responses

To test whether natural variation in Arabidopsis could be used to dissect out the genetic basis of responses to drought stress, we characterised a number of accessions. Most of the accessions belong to a core collection that was shown to maximise the genetic diversity captured for a given number of individual accessions in Arabidopsis thaliana. We measured total leaf area (TLA), Electrolyte Leakage (EL), Relative Water Content (RWC), and Cut Rosette Water Loss (CRWL) in control and mild water deficit conditions. A Principal Component Analysis revealed which traits explain most of the variation and showed that some accessions behave differently compared to the others in drought conditions, these included Ita-0, Cvi-0 and Shahdara. This study relied on genetic variation found naturally within the species, in which populations are assumed to be adapted to their environment. Overall, Arabidopsis thaliana showed interesting phenotypic variations in response to mild water deficit that can be exploited to identify genes and alleles important for this complex trait.     

Parasite local adaptation in a geographic mosaic

A central prediction of the geographic mosaic theory of coevolution is that coevolving interspecific interactions will show varying degrees of local maladaptation. According to the theory, much of this local maladaptation is driven by selection mosaics and spatially intermingled coevolutionary hot and cold spots, rather than a simple balance between gene flow and selection. Here I develop a genetic model of host-parasite coevolution that is sufficiently general to incorporate selection mosaics, coevolutionary hot and cold spots, and a diverse array of genetic systems of infection/resistance. Results from this model show that the selection mosaics experienced by the interacting species are an important determinant of the sign and magnitude of local maladaptation. In some cases, this effect may be stronger than a previously described effect of relative rates of parasite and host gene flow. These results provide the first theoretical evidence that selection mosaics and coevolutionary hot and cold spots per se determine the magnitude and sign of local maladaptation. At the same time, however, these results demonstrate that coevolution in a geographic mosaic can lead to virtually any pattern of local adaptation or local maladaptation. Consequently, empirical studies that describe only patterns of local adaptation or maladaptation do not provide evidence either for or against the theory.     

Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ～400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.     

Host-parasite interactions for virulence and resistance in a malaria model system

A rich body of theory on the evolution of virulence (disease severity) attempts to predict the conditions that cause parasites to harm their hosts, and a central assumption to many of these models is that the relative virulence of pathogen strains is stable across a range of host types. In contrast, a largely nonoverlapping body of theory on coevolution assumes that the fitness effects of parasites on hosts is not stable across host genotype, but instead depends on host genotype by parasite genotype interactions. If such genetic interactions largely determine virulence, it becomes difficult to predict the strength and direction of selection on virulence. In this study, we tested for host-by-parasite interactions in a medically relevant vertebrate disease model: the rodent malaria parasite Plasmodium chabaudi in laboratory mice. We found that parasite and particularly host main effects explained most of the variance in virulence (anaemia and weight loss), resistance (parasite burden) and transmission potential. Host-by-parasite interactions were of limited influence, but nevertheless had significant effects. This raises the possibility that host heterogeneity may affect the rate of any parasite response to selection on virulence. This study of rodent malaria is one of the first tests for host-by-parasite interactions in any vertebrate disease; host-by-parasite interactions typical of those assumed in coevolutionary models were present, but were by no means pervasive.     

Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus.

We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. ELI3 mRNA accumulates in A. thaliana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM1 locus, ELI3 mRNA accumulates to high levels 5-10 h post-inoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM1 action. The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h post-infiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the ELI3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm1 locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM1 using an assay generalizable to any pathogen induced plant defense phenomena.     

A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25 degrees C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.