Eric B. Holub

Intragenic Recombination and Diversifying Selection Contribute to the Evolution of Downy Mildew Resistance at the RPP8 Locus of Arabidopsis

Pathogen resistance (R) genes of the NBS-LRR class (for nucleotide binding site and leucine-rich repeat) are found in many plant species and confer resistance to a diverse spectrum of pathogens. Little is known about the mechanisms that drive NBS-LRR gene evolution in the host–pathogen arms race. We cloned the RPP8 gene (for resistance to Peronospora parasitica) and compared the structure of alleles at this locus in resistant Landsberg erecta (Ler-0) and susceptible Columbia (Col-0) accessions. RPP8-Ler encodes an NBS-LRR protein with a putative N-terminal leucine zipper and is more closely related to previously cloned R genes that confer resistance to bacterial pathogens than it is to other known RPP genes. The RPP8 haplotype in Ler-0 contains the functional RPP8-Ler gene and a nonfunctional homolog, RPH8A. In contrast, the rpp8 locus in Col-0 contains a single chimeric gene, which was likely derived from unequal crossing over between RPP8-Ler and RPH8A ancestors within a Ler-like haplotype. Sequence divergence among RPP8 family members has been accelerated by positive selection on the putative ligand binding region in the LRRs. These observations indicate that NBS-LRR molecular evolution is driven by the same mechanisms that promote rapid sequence diversification among other genes involved in non-self-recognition.

THREE GENES OF THE ARABIDOPSIS RPP1 COMPLEX RESISTANCE LOCUS RECOGNIZE DISTINCT PERONOSPORA PARASITICA AVIRULENCE DETERMINANTS

Plant resistance (R) genes have evolved specific recognition capabilities in defense against pathogens. The evolution of R gene function and maintenance of R gene diversity within a plant species are therefore of great interest. In the Arabidopsis accession Wassilewskija, the RPP1 region on chromosome 3 contains four genetically linked recognition specificities, conditioning resistance to different isolates of the biotrophic oomycete Peronospora parasitica (downy mildew). We show that three of four tightly linked genes in this region, designated RPP1-WsA, RPP1-WsB, and RPP1-WsC, encode functional products of the NBS-LRR (nucleotide binding site–leucine-rich repeat) R protein class. They possess a TIR (Toll, interleukin-1, resistance) domain that is characteristic of certain other NBS-LRR–type R proteins, but in addition, they have unique hydrophilic or hydrophobic N termini. Together, the three RPP1 genes account for the spectrum of resistance previously assigned to the RPP1 region and thus comprise a complex R locus. The distinct but partially overlapping resistance capabilities conferred by these genes are best explained by the hypothesis that each recognizes a different pathogen avirulence determinant. We present evidence suggesting that the RPP genes at this locus are subject to the same selective forces that have been demonstrated for structurally different LRR-type R genes.

A Mutation within the Leucine-Rich Repeat Domain of the Arabidopsis Disease Resistance Gene RPS5 Partially Suppresses Multiple Bacterial and Downy Mildew Resistance Genes

Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.

The Scale of Population Structure in Arabidopsis thaliana

The population structure of an organism reflects its evolutionary history and influences its evolutionary trajectory. It constrains the combination of genetic diversity and reveals patterns of past gene flow. Understanding it is a prerequisite for detecting genomic regions under selection, predicting the effect of population disturbances, or modeling gene flow. This paper examines the detailed global population structure of Arabidopsis thaliana. Using a set of 5,707 plants collected from around the globe and genotyped at 149 SNPs, we show that while A. thaliana as a species self-fertilizes 97% of the time, there is considerable variation among local groups. This level of outcrossing greatly limits observed heterozygosity but is sufficient to generate considerable local haplotypic diversity. We also find that in its native Eurasian range A. thaliana exhibits continuous isolation by distance at every geographic scale without natural breaks corresponding to classical notions of populations. By contrast, in North America, where it exists as an exotic species, A. thaliana exhibits little or no population structure at a continental scale but local isolation by distance that extends hundreds of km. This suggests a pattern for the development of isolation by distance that can establish itself shortly after an organism fills a new habitat range. It also raises questions about the general applicability of many standard population genetics models. Any model based on discrete clusters of interchangeable individuals will be an uneasy fit to organisms like A. thaliana which exhibit continuous isolation by distance on many scales.

Identification of R-Gene Homologous DNA Fragments Genetically Linked to Disease Resistance Loci in Arabidopsis thaliana

Disease resistance in plants is a desirable economic trait. A number of disease resistance genes from various plant species have been cloned so far. The gene products of some of these can be distinguished by the presence of an N-terminal nucleotide binding site and a C-terminal stretch of leucine-rich repeats. Although these gene products are structurally related, the DNA sequences are poorly conserved. Only parts of the nucleotide binding site share enough DNA identity to design primers for polymerase chain reaction amplification of related DNA sequences. Such primers were used to amplify different resistance-gene-like (RGL) DNA fragments from Arabidopsis thaliana accessions Landsberg erecta and Columbia. Almost all cloned DNA fragments were genetically closely linked with known disease resistance loci. Most RGL fragments were found in a clustered or dispersed multi-copy sequence organization, supporting the supposed correlation of RGL sequences and disease resistance loci.

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.

Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes

We describe the identification of a mutant in the Arabidopsis accession Columbia (Col-0) that exhibits enhanced downy mildew (edm1) susceptibility to several Peronospora parasitica isolates, including the RPP7-diagnostic isolate Hiks1. The mutation was mapped to chromosome IV and characterized physically as a 35-kb deletion spanning seven genes. One of these genes complemented the mutant to full wild-type resistance against all of the Peronospora isolates tested. This gene (AtSGT1b) encodes a predicted protein of 39.8 kD and is an Arabidopsis ortholog of yeast SGT1, which was described originally as a key regulatory protein in centromere function and ubiquitin-mediated proteolysis. AtSGT1b contains three tetratricopeptide repeats at the N terminus followed by a bipartite chord-containing SGT domain and an SGT-specific domain at the C terminus. We discuss the role of AtSGT1b in disease resistance and its possible involvement in ubiquitin-mediated proteolysis in plants.

Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana

Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires “effectors” to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the “CHXCs”, by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.

Genetic characterization of RRS1, a recessive locus in Arabidopsis thaliana that confers resistance to the bacterial soilborne pathogen Ralstonia solanacearum.

The soilborne, vascular pathogen Ralstonia solanacearum, the causative agent of bacterial wilt, was shown to infect a range of Arabidopsis thaliana accessions. The pathogen was capable of infecting the Col-5 accession in an hrp-dependent manner, following root inoculation. Elevated bacterial population levels were found in leaves of Col-5, 4 to 5 days after root inoculation by the GMI1000 strain. Bacteria were found predominantly in the xylem vessels and spread systematically throughout the plant. The Nd-1 accession of A. thaliana was resistant to the GMI1000 strain of R. solanacearum. Bacterial concentrations detected in leaves of Nd-1, inoculated with an hrp+ strain of R. solanacearum, were only slightly higher than those detected in the susceptible accession, Col-5, following inoculation with a strain whose hrp gene cluster was deleted. Leaf inoculation of the GMI1000 strain on the resistant accession Nd-1 induced the formation of lesions in the older leaves of the rosette whereas the same strain of R. solanacearum provoked complete wilting of Col-5. Resistance to strain GMI1000 of R. solanacearum segregated as a simply inherited recessive trait in a genetic cross between Col-5 and Nd-1. F9 recombinant inbred lines generated between these two accessions were used to map a locus, RRS1, that was the major determinant of resistance between restriction fragment length polymorphism markers mi83 and mi61 on chromosome V. This region of the A. thaliana genome is known to contain many other pathogen recognition capabilities.

MEDIATOR25 Acts as an Integrative Hub for the Regulation of Jasmonate-Responsive Gene Expression in Arabidopsis

The PHYTOCHROME AND FLOWERING TIME1 gene encoding the MEDIATOR25 (MED25) subunit of the eukaryotic Mediator complex is a positive regulator of jasmonate (JA)-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Based on the function of the Mediator complex as a bridge between DNA-bound transcriptional activators and the RNA polymerase II complex, MED25 has been hypothesized to function in association with transcriptional regulators of the JA pathway. However, it is currently not known mechanistically how MED25 functions to regulate JA-responsive gene expression. In this study, we show that MED25 physically interacts with several key transcriptional regulators of the JA signaling pathway, including the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 and ERF1 as well as the master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF transcription factors was shown to require the activator interaction domain of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-interacting AP2/ERFs. Using transcriptional activation experiments, we also show that OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59- and ERF1-dependent activation of PLANT DEFENSIN1.2 as well as MYC2-dependent activation of VEGETATIVE STORAGE PROTEIN1 requires a functional MED25. In addition, MED25 is required for MYC2-dependent repression of pathogen defense genes. These results suggest an important role for MED25 as an integrative hub within the Mediator complex during the regulation of JA-associated gene expression.