Recent emergence of the wheat Lr34 multi-pathogen resistance: insights from haplotype analysis in wheat, rice, sorghum and Aegilops tauschii

Spontaneous sequence changes and the selection of beneficial mutations are driving forces of gene diversification and key factors of evolution. In highly dynamic co-evolutionary processes such as plant-pathogen interactions, the plant's ability to rapidly adapt to newly emerging pathogens is paramount. The hexaploid wheat gene Lr34, which encodes an ATP-binding cassette (ABC) transporter, confers durable field resistance against four fungal diseases. Despite its extensive use in breeding and agriculture, no increase in virulence towards Lr34 has been described over the last century. The wheat genepool contains two predominant Lr34 alleles of which only one confers disease resistance. The two alleles, located on chromosome 7DS, differ by only two exon-polymorphisms. Putatively functional homoeologs and orthologs of Lr34 are found on the B-genome of wheat and in rice and sorghum, but not in maize, barley and Brachypodium. In this study we present a detailed haplotype analysis of homoeologous and orthologous Lr34 genes in genetically and geographically diverse selections of wheat, rice and sorghum accessions. We found that the resistant Lr34 haplotype is unique to the wheat D-genome and is not found in the B-genome of wheat or in rice and sorghum. Furthermore, we only found the susceptible Lr34 allele in a set of 252 Ae. tauschii genotypes, the progenitor of the wheat D-genome. These data provide compelling evidence that the Lr34 multi-pathogen resistance is the result of recent gene diversification occurring after the formation of hexaploid wheat about 8,000 years ago.

Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids.

Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen.

The role of Di-iron proteins in pathogen resistance

Dissertation presented to obtain the Ph.D. degree in Biochemistry

Volatiles produced by soil-borne endophytic bacteria increase plant pathogen resistance and affect tritrophic interactions

Volatile organic compounds (VOCs) released by soil microorganisms influence plant growth and pathogen resistance. Yet, very little is known about their influence on herbivores and higher trophic levels. We studied the origin and role of a major bacterial VOC, 2,3-butanediol (2,3-BD), on plant growth, pathogen and herbivore resistance, and the attraction of natural enemies in maize. One of the major contributors to 2,3-BD in the headspace of soil-grown maize seedlings was identified as Enterobacter aerogenes, an endophytic bacterium that colonizes the plants. The production of 2,3-BD by E. aerogenes rendered maize plants more resistant against the Northern corn leaf blight fungus Setosphaeria turcica. On the contrary, E. aerogenes-inoculated plants were less resistant against the caterpillar Spodoptera littoralis. The effect of 2,3-BD on the attraction of the parasitoid Cotesia marginiventris was more variable: 2,3-BD application to the headspace of the plants had no effect on the parasitoids, but application to the soil increased parasitoid attraction. Furthermore, inoculation of seeds with E. aerogenes decreased plant attractiveness, whereas inoculation of soil with a total extract of soil microbes increased parasitoid attraction, suggesting that the effect of 2,3-BD on the parasitoid is indirect and depends on the composition of the microbial community.

Synergies and trade-offs between insect and pathogen resistance in maize leaves and roots

Determining links between plant defence strategies is important to understand plant evolution and to optimize crop breeding strategies. Although several examples of synergies and trade-offs between defence traits are known for plants that are under attack by multiple organisms, few studies have attempted to measure correlations of defensive strategies using specific single attackers. Such links are hard to detect in natural populations because they are inherently confounded by the evolutionary history of different ecotypes. We therefore used a range of 20 maize inbred lines with considerable differences in resistance traits to determine if correlations exist between leaf and root resistance against pathogens and insects. Aboveground resistance against insects was positively correlated with the plant's capacity to produce volatiles in response to insect attack. Resistance to herbivores and resistance to a pathogen, on the other hand, were negatively correlated. Our results also give first insights into the intraspecific variability of root volatiles release in maize and its positive correlation with leaf volatile production. We show that the breeding history of the different genotypes (dent versus flint) has influenced several defensive parameters. Taken together, our study demonstrates the importance of genetically determined synergies and trade-offs for plant resistance against insects and pathogens.

Impact of thermal stress on evolutionary trajectories of pathogen resistance in three-spined stickleback (Gasterosteus aculeatus), supplementary material

Background: Pathogens are a major regulatory force for host populations, especially under stressful conditions. Elevated temperatures may enhance the development of pathogens, increase the number of transmission stages, and can negatively influence host susceptibility depending on host thermal tolerance. As a net result, this can lead to a higher prevalence of epidemics during summer months. These conditions also apply to marine ecosystems, where possible ecological impacts and the population-specific potential for evolutionary responses to changing environments and increasing disease prevalence are, however, less known. Therefore, we investigated the influence of thermal stress on the evolutionary trajectories of disease resistance in three marine populations of three-spined sticklebacks Gasterosteus aculeatus by combining the effects of elevated temperature and infection with a bacterial strain of Vibrio sp. using a common garden experiment. Results: We found that thermal stress had an impact on fish weight and especially on survival after infection after only short periods of thermal acclimation. Environmental stress reduced genetic differentiation (QST) between populations by releasing cryptic within-population variation. While life history traits displayed positive genetic correlations across environments with relatively weak genotype by environment interactions (GxE), environmental stress led to negative genetic correlations across environments in pathogen resistance. This reversal of genetic effects governing resistance is probably attributable to changing environment-dependent virulence mechanisms of the pathogen interacting differently with host genotypes, i.e. GPathogenxGHostxE or (GPathogenxE)x(GHostxE) interactions, rather than to pure host genetic effects, i.e. GHostxE interactions. Conclusion: To cope with climatic changes and the associated increase in pathogen virulence, host species require wide thermal tolerances and pathogen-resistant genotypes. The higher resistance we found for some families at elevated temperatures showed that there is evolutionary potential for resistance to Vibrio sp. in both thermal environments. The negative genetic correlation of pathogen resistance between thermal environments, on the other hand, indicates that adaptation to current conditions can be a weak predictor for performance in changing environments. The observed feedback on selective gradients exerted on life history traits may exacerbate this effect, as it can also modify the response to selection for other vital components of fitness.

Independent deletions of a pathogen-resistance gene in Brassica and Arabidopsis

Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with ≈81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.

UV induces resistance in Arabidopsis Thaliana to the Oomycete Pathogen Hyaloperonospora Parasitica

Owing to their sessile nature, plants have evolved mechanisms to minimise the damaging effects of abiotic and biotic stresses. Attack by pathogenic fungi, viruses and bacterium is a major type of biotic stress. To resist infection, plants recognise invading pathogens and induce disease resistance through multiple signal transduction pathways. In addition, appropriate stimulation can cause plants to increase their resistance to future pathogen attack. We have found that exposure to non-lethal doses of UV-C (254 nm) renders a normally susceptible ecotype of Arabidopsis thaliana resistant to the biotrophic Oomycete pathogen Hyaloperonospora parasitica. The UV treatment induces an incompatible response in a dose-dependent fashion, and is still effective upon pathogen inoculation up to seven days after UV exposure. The degree of resistance diminishes with time but higher doses result in greater levels of resistance, even after seven days. Furthermore, the effect is systemic, occurring in parts of the plant that have not been irradiated. Incubation in the dark post?irradiation and prior to infection reduces the UV dose required to generate a specific level of pathogen resistance without affecting the duration of resistance. These observations, plus the inability of plants to photoreactivate UV photoproducts in the dark, strongly suggest that DNA damage induces the resistance phenotype. Currently, we are assessing the influence of DNA repair defects on UV-induced resistance, following the expression of a number of defence?related genes post-UV-C irradiation, and assessing the effect of UV in plant mutants deficient in specific signalling molecules involved in resistance.

UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

Two distinct classes of QTL determine rust resistance in sorghum

Background: Agriculture is facing enormous challenges to feed a growing population in the face of rapidly evolving pests and pathogens. The rusts, in particular, are a major pathogen of cereal crops with the potential to cause large reductions in yield. Improving stable disease resistance is an on-going major and challenging focus for many plant breeding programs, due to the rapidly evolving nature of the pathogen. Sorghum is a major summer cereal crop that is also a host for a rust pathogen which occurs in almost all sorghum growing areas of the world, causing direct and indirect yield losses in sorghum worldwide, however knowledge about its genetic control is still limited. In order to further investigate this issue, QTL and association mapping methods were implemented to study rust resistance in three bi-parental populations and an association mapping set of elite breeding lines in different environments. Results: In total, 64 significant or highly significant QTL and 21 suggestive rust resistance QTL were identified representing 55 unique genomic regions. Comparisons across populations within the current study and with rust QTL identified previously in both sorghum and maize revealed a high degree of correspondence in QTL location. Negative phenotypic correlations were observed between rust, maturity and height, indicating a trend for both early maturing and shorter genotypes to be more susceptible to rust. Conclusions: The significant amount of QTL co-location across traits, in addition to the consistency in the direction of QTL allele effects, has provided evidence to support pleiotropic QTL action across rust, height, maturity and stay-green, supporting the role of carbon stress in susceptibility to rust. Classical rust resistance QTL regions that did not co-locate with height, maturity or stay-green QTL were found to be significantly enriched for the defence-related NBS-encoding gene family, in contrast to the lack of defence-related gene enrichment in multi-trait effect rust resistance QTL. The distinction of disease resistance QTL hot-spots, enriched with defence-related gene families from QTL which impact on development and partitioning, provides plant breeders with knowledge which will allow for fast-tracking varieties with both durable pathogen resistance and appropriate adaptive traits.