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Mining disease-resistance genes in roses: functional and molecular characterization of the rdr1 locus.

ABSTRACT: The interaction of roses with the leaf spot pathogen Diplocarpon rosae (the cause of black spot on roses) is an interesting pathosystem because it involves a long-lived woody perennial, with life history traits very different from most model plants, and a hemibiotrophic pathogen with moderate levels of gene flow. Here we present data on the molecular structure of the first monogenic dominant resistance gene from roses, Rdr1, directed against one isolate of D. rosae. Complete sequencing of the locus carrying the Rdr1 gene resulted in a sequence of 265,477?bp with a cluster of nine highly related TIR-NBS-LRR (TNL) candidate genes. After sequencing revealed candidate genes for Rdr1, we implemented a gene expression analysis and selected five genes out of the nine TNLs. We then silenced the whole TNL gene family using RNAi (Rdr1-RNAi) constructed from the most conserved sequence region and demonstrated a loss of resistance in the normally resistant genotype. To identify the functional TNL gene, we further screened the five TNL candidate genes with a transient leaf infiltration assay. The transient expression assay indicated a single TNL gene (muRdr1H), partially restoring resistance in the susceptible genotype. Rdr1 was found to localize within the muRdr1 gene family; the genes within this locus contain characteristic motifs of active TNL genes and belong to a young cluster of R genes. The transient leaf assay can be used to further analyze the rose black spot interaction and its evolution, extending the analyses to additional R genes and to additional pathogenic types of the pathogen.

SUBMITTER: Terefe-Ayana D

PROVIDER: S-EPMC3355636 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Mining disease-resistance genes in roses: functional and molecular characterization of the rdr1 locus.

Terefe-Ayana Diro D Yasmin Aneela A Le Thanh Loan TL Kaufmann Helgard H Biber Anja A Kühr Astrid A Linde Marcus M Debener Thomas T

Frontiers in plant science 20110801

The interaction of roses with the leaf spot pathogen Diplocarpon rosae (the cause of black spot on roses) is an interesting pathosystem because it involves a long-lived woody perennial, with life history traits very different from most model plants, and a hemibiotrophic pathogen with moderate levels of gene flow. Here we present data on the molecular structure of the first monogenic dominant resistance gene from roses, Rdr1, directed against one isolate of D. rosae. Complete sequencing of the lo ...[more]

PMID: 22639591

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Project description:BackgroundThe resistance of plants to pathogens relies on two lines of defense: a basal defense response and a pathogen-specific system, in which resistance (R) genes induce defense reactions after detection of pathogen-associated molecular patterns (PAMPS). In the specific system, a so-called arms race has developed in which the emergence of new races of a pathogen leads to the diversification of plant resistance genes to counteract the pathogens' effect. The mechanism of resistance gene diversification has been elucidated well for short-lived annual species, but data are mostly lacking for long-lived perennial and clonally propagated plants, such as roses. We analyzed the rose black spot resistance gene, Rdr1, in five members of the Rosaceae: Rosa multiflora, Rosa rugosa, Fragaria vesca (strawberry), Malus x domestica (apple) and Prunus persica (peach), and we present the deduced possible mechanism of R-gene diversification.ResultsWe sequenced a 340.4-kb region from R. rugosa orthologous to the Rdr1 locus in R. multiflora. Apart from some deletions and rearrangements, the two loci display a high degree of synteny. Additionally, less pronounced synteny is found with an orthologous locus in strawberry but is absent in peach and apple, where genes from the Rdr1 locus are distributed on two different chromosomes. An analysis of 20 TIR-NBS-LRR (TNL) genes obtained from R. rugosa and R. multiflora revealed illegitimate recombination, gene conversion, unequal crossing over, indels, point mutations and transposable elements as mechanisms of diversification.A phylogenetic analysis of 53 complete TNL genes from the five Rosaceae species revealed that with the exception of some genes from apple and peach, most of the genes occur in species-specific clusters, indicating that recent TNL gene diversification began prior to the split of Rosa from Fragaria in the Rosoideae and peach from apple in the Spiraeoideae and continued after the split in individual species. Sequence similarity of up to 99% is obtained between two R. multiflora TNL paralogs, indicating a very recent duplication.ConclusionsThe mechanisms by which TNL genes from perennial Rosaceae diversify are mainly similar to those from annual plant species. However, most TNL genes appear to be of recent origin, likely due to recent duplications, supporting the hypothesis that TNL genes in woody perennials are generally younger than those from annuals. This recent origin might facilitate the development of new resistance specificities, compensating for longer generation times in woody perennials.

Project description:A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the ?1 subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide/cGMP signaling pathway. The mechanism linking common variants in this region with coronary risk is not known.Gene expression and protein expression were analyzed with quantitative polymerase chain reaction and immunoblotting, respectively. Putative allele-specific transcription factors were identified with in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed with reporter gene assays. To assess the effect of zinc finger E box-binding homeobox 1 transcription factor (ZEB1), siRNA-mediated knockdown and overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes was analyzed with vascular smooth muscle cell migration assays and platelet aggregation analyses.Whole-blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the nonrisk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of nonrisk allele promoter activity and a significant reduction of endogenous GUCY1A3 expression. Ex vivo-studied platelets from homozygous nonrisk allele carriers displayed enhanced inhibition of ADP-induced platelet aggregation by the nitric oxide donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil compared with homozygous risk allele carriers. Moreover, pharmacological stimulation of sGC led to reduced migration only in vascular smooth muscle cells homozygous for the nonrisk allele. In the Hybrid Mouse Diversity Panel, higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta.Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the nonrisk allele, leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.

Project description:Glutamate-like receptor (GLR) genes are a group of regulatory genes involved in many physiological processes of plants. With 26 members in the rice genome, the functionalities of most rice GLR genes remain unknown. To facilitate their potential uses in rice improvement, an integrated strategy involving CRISPR-Cas9 mediated knockouts, deep mining and analyses of transcriptomic responses to different abiotic stresses/hormone treatments and gene CDS haplotype (gcHap) diversity in 3,010 rice genomes was taken to understand the functionalities of the 26 rice GLR genes, which led us to two conclusions. First, the expansion of rice GLR genes into a large gene family during evolution had gone through repeated gene duplication events occurred primarily in two large GLR gene clusters on rice chromosomes 9 and 6, which was accompanied with considerable functional differentiation. Secondly, except for two extremely conserved ones (OsGLR6.2 and OsGLR6.3), rich gcHap diversity exists at the remaining GLR genes which played important roles in rice population differentiation and rice improvement, evidenced by their very strong sub-specific and population differentiation, by their differentiated responses to day-length and different abiotic stresses, by the large phenotypic effects of five GLR gene knockout mutants on rice yield traits, by the significant association of major gcHaps at most GLR loci with yield traits, and by the strong genetic bottleneck effects and artificial selection on the gcHap diversity in populations Xian (indica) and Geng (japonica) during modern breeding. Our results suggest the potential values of the natural variation at most rice GLR loci for improving the productivity and tolerances to abiotic stresses. Additional efforts are needed to determine the phenotypic effects of major gcHaps at these GLR loci in order to identify 'favorable' alleles at specific GLR loci specific target traits in specific environments to facilitate their application to rice improvement in future.

Dataset Information

Mining disease-resistance genes in roses: functional and molecular characterization of the rdr1 locus.

Publications

Mining disease-resistance genes in roses: functional and molecular characterization of the rdr1 locus.

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