Disease resistance gene homologs correlate with disease resistance loci of Arabidopsis thaliana

The disease resistance genes RPS2 of Arabidopsis and N of tobacco, among other recently cloned resistance genes, share several conserved sequences. Degenerate oligonucleotide primers, based on conserved sequences in the nucleotide binding site (NBS) and a weak hydrophobic domain of RPS2 and N, were used to amplify homologous sequences from Arabidopsis thaliana. Amplification products were obtained that were similar in sequence to the disease resistance genes RPS2, RPM1, N and L6. The Arabidopsis CIC-YAC library was used to identify the position of the disease resistance homologs on the Arabidopsis genome. Their map positions could be correlated with the disease resistance loci RPS5, RAC1, RPP9, CAR1, RPP7, RPW2, RPP1, RPP10, RPP14, RPP5, RPP4, RPS2, RPW6, HRT, RPS4, RPP8, RPP21, RPP22, RPP23, RPP24 and TTR1. This method was therefore not only successful in the identification of sequences located within gene clusters that are involved in disease resistance, but could also contribute to the cloning of disease resistance genes from Arabidopsis.     

Functional variation in a disease resistance gene in populations of Arabidopsis thaliana

Analyses of functional genetic diversity in natural populations may provide important new insights into gene function and are necessary to understand the evolutionary processes maintaining diversity itself. The importance of including diversity within and between local populations in such studies is often ignored although many of the processes affecting genetic diversity act on this scale. Here we examine the molecular diversity in RPW8 (Recognition of Powdery Mildew), a gene conferring broad‐spectrum resistance to powdery mildews in Arabidopsis thaliana stock‐center accessions. Our eight UK study populations of the weedy A. thaliana were from locations judged to be subject to a minimum of anthropogenic disturbance and potentially long established. The majority of populations comprised considerable variation both in disease phenotype and RPW8 genotype. Although resistant individuals shared a major RPW8 genotype, no single allele was uniquely associated with resistance. It is concluded that RPW8 is an essential component of resistance to powdery mildews in A. thaliana, but not the only genetic factor involved in this process. No signature of selection was detected at RPW8 with a microsatellite multilocus test using an empirical null model. Unlike many previous studies of this model plant species, we found high levels of genetic diversity and relatively low differentiation (F_ST = 0.31) between populations at 14 microsatellite markers. This is judged to be due to our sampling being aimed at potentially long established populations and highlights the importance of population choice for studies of genetic diversity within this species.     

A non-destructive selection method for resistance to fusicoccin in Arabidopsis thaliana

A mutagenised population of seeds of Arabidopsis thaliana was allowed to germinate in the presence of the positively charged aminoglycoside hygromycin (4 μg/ml) and the fungal toxin fusicoccin (5×10^–6 m). This hygromycin concentration, which is non-toxic by itself, becomes toxic when used together with fusicoccin, which stimulates cation uptake. Seeds that had germinated after 3–5 days and produced seedlings with green cotyledons were potentially resistant to fusicoccin and were therefore transferred into sterile Magenta vessels containing 1/2-strength Murashige and Skoog medium. This selection procedure is non-destructive, i.e. it allows the recovery of viable seedlings and their growth into adult plants thus permitting direct physiological characterisation. Received: 16 February 1998 / Revision received: 11 August 1998 / Accepted: 13 August 1998     

Variation and phylogenetic utility of the Arabidopsis thaliana Rps2 homolog in various species of the tribe Brassiceae

Our objective was to analyze the evolutionary paths of cultivated diploid Brassica species and a few related wild species (tribe Brassiceae) in relation to Arabidopsis thaliana (tribe Arabidae), using the Rps2 sequence. Rps2 confers resistance to Pseudomonas syringae in A. thaliana. We found that similar to Arabidopsis, the Rps2 homolog in Brassica species is present in a single copy. Primers based in the Rps2 sequence amplified Rps2 homologs from the other species. Maximum-parsimony analysis based on number of nucleotide substitutions yielded a single tree, grouping the species as expected from other evolutionary inferences. Age of divergence between the two tribes was within the range of previous estimates. Indels in the different sequences were also useful for distinguishing some of the species. The Rps2 gene is a useful phylogenetic tool for more comprehensive studies of the species of Brasicaceae.     

DNA polymorphism at the FRIGIDA gene in Arabidopsis thaliana: extensive nonsynonymous variation is consistent with local selection for flowering time

FRIGIDA (FRI) is a major gene involved in the regulation of flowering time in Arabidopsis thaliana. Nucleotide variation at this gene was investigated by sequencing 25 field ecotypes collected from western Europe. Genetic diversity at FRI was characterized by a high number of haplotypes and an excess of low-frequency polymorphisms. A large excess of intraspecific nonsynonymous variation associated with low synonymous variation was detected along the first exon in the FRI gene. In contrast, no excess of nonsynonymous divergence was detected between A. thaliana and A. lyrata. The Tajima and McDonald and Kreitman tests, however, suggested that this gene has evolved in a nonneutral fashion. Nonsynonymous variation included eight loss-of-function mutations that have probably arisen recently and independently in several locations. A phenotypic evaluation of the sequenced ecotypes confirmed that these loss-of-function mutations were associated with an early-flowering phenotype. Taken together, our results suggest that DNA polymorphism at the FRI gene in A. thaliana from western Europe has been shaped by recent positive selection for earliness in a set of isolated populations.     

The pattern of polymorphism in Arabidopsis thaliana

We resequenced 876 short fragments in a sample of 96 individuals of Arabidopsis thaliana that included stock center accessions as well as a hierarchical sample from natural populations. Although A. thaliana is a selfing weed, the pattern of polymorphism in general agrees with what is expected for a widely distributed, sexually reproducing species. Linkage disequilibrium decays rapidly, within 50 kb. Variation is shared worldwide, although population structure and isolation by distance are evident. The data fail to fit standard neutral models in several ways. There is a genome-wide excess of rare alleles, at least partially due to selection. There is too much variation between genomic regions in the level of polymorphism. The local level of polymorphism is negatively correlated with gene density and positively correlated with segmental duplications. Because the data do not fit theoretical null distributions, attempts to infer natural selection from polymorphism data will require genome-wide surveys of polymorphism in order to identify anomalous regions. Despite this, our data support the utility of A. thaliana as a model for evolutionary functional genomics.     

Soybean phytophthora resistance gene Rps8 maps closely to the Rps3 region

Root and stem rot is one of the major diseases of soybean. It is caused by the oomycete pathogen Phytophthora sojae. A series of resistance genes (Rps) have been providing soybean with reasonable protection against this pathogen. Among these genes, Rps8, which confers resistance to most P. sojae isolates, recently has been mapped. However, the most closely linked molecular marker was mapped at about 10 cM from Rps8. In this investigation, we attempted to develop a high-density genetic map of the Rps8 region and identify closely linked SSR markers for marker-assisted selection of this invaluable gene. Bulk segregant analysis was conducted for the identification of SSR markers that are tightly linked to Rps8. Polymorphic SSR markers selected from the Rps8 region failed to show cosegregation with Phytophthora resistance. Subsequently, bulk segregant analysis of the whole soybean genome and mapping experiments revealed that the Rps8 gene maps closely to the disease resistance gene-rich Rps3 region.     

Natural variation for seed oil composition in Arabidopsis thaliana

The biochemical pathways involved in the biosynthesis and accumulation of storage lipids in seeds have been extensively studied. However, the regulatory mechanisms of those pathways, their environmental interactions and the ecological implications of variation are poorly understood. We have initiated a new approach: the analysis of natural variation in Arabidopsis thaliana. Three hundred and sixty accessions were surveyed for content of oil, very long chain fatty acids (VLCFAs) and polyunsaturated fatty acids (PUFAs) in their seeds. The results revealed extensive natural variation. A core set of accessions, the seeds of which reproducibly contain extreme amounts of oil, VLCFAs and PUFAs have been identified. Reproducible oil content ranged from 34.6 to 46.0% of seed dry weight. VLCFA content ranged from 13.0 to 21.2% of total fatty acids. PUFA content, ranged from 53.3 to 66.1% of total fatty acids. Interactions were also identified for PUFA and VLCFA content of seeds with vernalisation of plants. Mapping of the regions of the genome involved in controlling the traits was conducted in an F(2) population and indicated that natural variation at the loci FAE1 and FAD3 might be involved in the regulation of VLCFA and PUFA content, respectively. A set of accessions, which capture a broad range of the natural variation for these traits available in A. thaliana, has been selected to form a core set which can be used to further dissect the genetics of the regulation of seed lipid traits and to identify the genes involved.     

天山北麓拟南芥表型特征的自然选择

本文研究了天山北麓干旱、湿润2种不同水分条件下拟南芥(Arabidopsis thaliana)光合生理、形态及发育特征的自然适应特点。结果表明：分枝数、株高、相对适合度（角果质量）、莲座叶面积等特征在不同水分下差异极显著;根质量与相对适合度、莲座叶面积、水分利用效率和光合速率之间表现了正相关,胞间二氧化碳浓度与光合速率、水分利用效率之间表现了负相关;选择梯度分析得出,干湿水分条件下分枝数、株高、莲座叶面积均表现定向选择;主成分分析发现,光合生理特征整合在处理间有一定的规律性,表现出拟南芥物种特征整体协调一致性较强,对环境响应具有较大的趋同性;通径分析发现,水分利用效率、枝质量、株高、角果数对相对适合度直接产生较大影响,因而在通径模型中设置为直接路径特征,将莲座叶面积通过分枝数、花期、角果数,花期通过角果数到达相对适合度的过程设置为间接路径,这些特征通过间接路径对相对适合度间接产生较大影响。     

Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana

This paper reports on six Arabidopsis accessions that show resistance to a wild isolate of the powdery mildew pathogen, Erysiphe cichoracearum . Resistance at 7 days post-inoculation in these accessions was characterized by limited fungal growth and sporadic development of chlorotic or necrotic lesions at inoculation sites. Three accessions, Wa-1, Kas-1 and SI-0, were highly resistant, while the other accessions permitted some fungal growth and conidiation. Papilla formation was a frequent host response; however, cell death appeared to be neither a rapid nor a common response to infection. To determine the genetic basis of resistance, segregation analyses of progeny from crosses between each of the resistant accessions and Columbia ( gl1 ), which is susceptible to the powdery mildew pathogen, were performed. For all accessions except SI-0, resistance was conferred by a single locus. SI-0 was unique in that two unlinked loci controlled the disease reaction phenotype. In accessions Wa-1, Kas-1, Stw-0 and Su-0, powdery mildew resistance was encoded by a semi-dominant allele. However, susceptibility was dominant to resistance in accessions Te-0 and SI-0. Mapping studies revealed that powdery mildew resistances in Kas-1, Wa-1, Te-0, Su-0 and Stw-0 were controlled by five independent loci. This study suggests that the Arabidopsis powdery mildew disease will be a suitable model system in which to investigate powdery mildew diseases.     

Nucleotide polymorphism at the Atmyb2 locus of the wild plant Arabidopsis thaliana

DNA variation was studied in a 2.2 kb region of the regulatory gene Atmyb2 using 20 ecotypes of Arabidopsis thaliana and one accession each of Arabis gemmifera and Arabidopsis himalaica. Nucleotide diversity (pi) in the region was 0.0027, which was lower than for other loci in A. thaliana. The MYB domain of the Atmyb2 gene (pi = 0.0036) had a larger variation than the non-MYB region (pi = 0.0013). Tajima's test and Fu and Li's test did not give a significant result. In contrast to the low level of polymorphism, the degree of divergence of the Atmyb2 region was higher between A. thaliana and A. gemmifera (K = 0.0730) than for other loci. The MYB domain (K = 0.0436) had smaller divergence than the non-MYB region (K = 0.0939). The HKA test detected significant discordance in the ratio of polymorphism to divergence in some comparisons. The pattern of low polymorphism and high divergence, which is mainly observed in the non-MYB region of the gene, is inconsistent with the neutral mutation theory. Strong purifying selection after establishment of A. thaliana and a species-specific adaptive process could be invoked to account for this pattern of polymorphism and divergence of Atmyb2.     

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.     

Phospholipase-dependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana

Bacterial pathogens deliver type III effector proteins into plant cells during infection. On susceptible host plants, type III effectors contribute to virulence, but on resistant hosts they betray the pathogen to the plant's immune system and are functionally termed avirulence (Avr) proteins. Recognition induces a complex suite of cellular and molecular events comprising the plant's inducible defence response. As recognition of type III effector proteins occurs inside host cells, defence responses can be elicited by in planta expression of bacterial type III effectors. We demonstrate that recognition of either of two type III effectors, AvrRpm1 or AvrRpt2 from Pseudomonas syringae , induced biphasic accumulation of phosphatidic acid (PA). The first wave of PA accumulation correlated with disappearance of monophosphatidylinosotol (PIP) and is thus tentatively attributed to activation of a PIP specific phospholipase C (PLC) in concert with diacylglycerol kinase (DAGK) activity. Subsequent activation of phospholipase D (PLD) produced large amounts of PA from structural phospholipids. This later wave of PA accumulation was several orders of magnitude higher than the PLC-dependent first wave. Inhibition of phospholipases blocked the response, and feeding PA directly to leaf tissue caused cell death and defence-gene activation. Inhibitor studies ordered these events relative to other known signalling events during the plant defence response. Influx of extracellular Ca²⁺ occurred downstream of PIP-degradation, but upstream of PLD activation. Production of reactive oxygen species occurred downstream of the phospholipases. The data presented indicate that PA is a positive regulator of RPM1- or RPS2-mediated disease resistance signalling, and that the biphasic PA production may be a conserved feature of signalling induced by the coiled-coil nucleotide binding domain leucine-rich repeat class of resistance proteins.     

Natural variation in the Arabidopsis response to the avirulence gene hopPsyA uncouples the hypersensitive response from disease resistance

The plant hypersensitive response (HR) is tightly associated with gene-for-gene resistance and has been proposed to function in containing pathogens at the invasion site. This tight association has made it difficult to unequivocally evaluate the importance of HR for plant disease resistance. Here, hopPsyA from Pseudomonas syringae pv. syringae 61 is identified as a new avirulence gene for Arabidopsis that triggers resistance in the absence of macroscopic HR. Resistance to P. syringae pv. tomato DC3000 expressing hopPsyA was EDS1-dependent and NDR1-independent. Intriguingly, several Arabidopsis accessions were resistant to DC3000(hopPsyA) in the absence of HR. This is comparable to the Arabidopsis response to avrRps4, but it is shown that hopPsyA does not signal through RPS4. In a cross between two hopPsyA-resistant accessions that differ in their HR response, the HR segregated as a recessive phenotype regulated by a single locus. This locus, HED1 (HR regulator in EDS1 pathway), is proposed to encode a protein whose activity can cause suppression of the EDS1-dependent HR signaling pathway. HED1-regulated symptomless gene-for-gene resistance responses may explain some cases of Arabidopsis resistance to bacteria that are classified as nonhost resistance.     

Genome-level evolution of resistance genes in Arabidopsis thaliana

Pathogen resistance genes represent some of the most abundant and diverse gene families found within plant genomes. However, evolutionary mechanisms generating resistance gene diversity at the genome level are not well understood. We used the complete Arabidopsis thaliana genome sequence to show that most duplication of individual NBS-LRR sequences occurs at close physical proximity to the parent sequence and generates clusters of closely related NBS-LRR sequences. Deploying the statistical strength of phylogeographic approaches and using chromosomal location as a proxy for spatial location, we show that apparent duplication of NBS-LRR genes to ectopic chromosomal locations is largely the consequence of segmental chromosome duplication and rearrangement, rather than the independent duplication of individual sequences. Although accounting for a smaller fraction of NBS-LRR gene duplications, segmental chromosome duplication and rearrangement events have a large impact on the evolution of this multigene family. Intergenic exchange is dramatically lower between NBS-LRR sequences located in different chromosome regions as compared to exchange between sequences within the same chromosome region. Consequently, once translocated to new chromosome locations, NBS-LRR gene copies have a greater likelihood of escaping intergenic exchange and adopting new functions than do gene copies located within the same chromosomal region. We propose an evolutionary model that relates processes of genome evolution to mechanisms of evolution for the large, diverse, NBS-LRR gene family.