The Avr1b locus of Phytophthora sojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b

We have used map-based approaches to clone a locus containing two genes, Avr1b-1 and Avr1b-2, required for avirulence of the oomycete pathogen Phytophthora sojae (Kaufmann & Gerdemann) on soybean plants carrying resistance gene Rps1b. Avr1b-1 was localized to a single 60-kb bacterial artificial chromosome (BAC) clone by fine-structure genetic mapping. Avr1b-1 was localized within the 60-kb region by identification of an mRNA that is expressed in a race-specific and infection-specific manner and that encodes a small secreted protein. When the Avr1b-1 protein was synthesized in the yeast Pichia pastoris and the secreted protein infiltrated into soybean leaves, it triggered a hypersensitive response specifically in host plants carrying the Rps1b resistance gene. This response eventually spread to the entire inoculated plant. In some isolates of P. sojae virulent on Rps1b-containing cultivars, such as P7081 (race 25) and P7076 (race 19), the Avr1b-1 gene had numerous substitution mutations indicative of strong divergent selection. In other isolates, such as P6497 (race 2) and P9073 (race 25), there were no substitutions in Avr1b-1, but Avr1b-1 mRNA did not accumulate. Genetic complementation experiments with P6497 revealed the presence of a second gene, Avr1b-2, required for the accumulation of Avr1b-1 mRNA. Avr1b-2 was genetically mapped to the same BAC contig as Avr1b-1, using a cross between P7064 (race 7) and P6497. The Avr1k gene, required for avirulence on soybean cultivars containing Rps1k, was mapped to the same interval as Avr1b-1.     

Genetic and physical mapping of Avr1a in Phytophthora sojae

The interaction between soybean and the phytopathogenic oomycete Phytophthora sojae is controlled by host resistance (Rps) genes and pathogen avirulence (Avr) genes. We have mapped the Avr1a locus in F(2) populations derived from four different P. sojae races. Four RAPD and nine AFLP markers linked to Avr1a were initially identified. Nine markers were used to compare genetic linkage maps of the Avr1a locus in two distinct F(2) populations. Distorted segregation ratios favoring homozygous genotypes were noted in both crosses. Segregation analysis of all the markers in one F(2) population of 90 progeny generated a map of 113.2 cM encompassing Avr1a, with one marker cosegregating with the gene. The cosegregating DNA marker was used to isolate P. sojae BAC clones and construct a physical map covering 170 kb, from which additional DNA markers were developed. Three markers occurring within the BAC contig were mapped in an enlarged population of 486 F(2) progeny. Avr1a was localized to a 114-kb interval, and an average physical to genetic distance ratio of 391 kb/cM was calculated for this region. This work provides a basis for the positional cloning of Avr1a.     

Salicylic acid-dependent expression of host genes in compatible Arabidopsis-virus interactions

Plant viruses elicit the expression of common sets of genes in susceptible hosts. Studies in Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) indicate that at least one-third of the genes induced in common by viruses have been previously associated with plant defense and stress responses. The genetic and molecular requirements for the induction of these stress and defense-related genes during compatible host-virus interactions were investigated with a panel of Arabidopsis mutant and transgenic plants defective in one or more defense signaling pathways. pad4, eds5, NahG, npr1, jar1, ein2, sid2, eds1, and wild-type Columbia-0 and Wassilewskija-2 plants were infected with two different viruses, cucumber mosaic virus and oilseed rape mosaic virus. Gene expression was assayed by a high-throughput fiber-optic bead array consisting of 388 genes and by RNA gel blots. These analyses demonstrated that, in compatible host-virus interactions, the expression of the majority of defense-related genes is induced by a salicylic acid-dependent, NPR1-independent signaling pathway with a few notable exceptions that did require NPR1. Interestingly, none of the mutant or transgenic plants showed enhanced susceptibility to either cucumber mosaic virus or oilseed rape mosaic virus based on both symptoms and virus accumulation. This observation is in contrast to the enhanced disease susceptibility phenotypes that these mutations or transgenes confer to some bacterial and fungal pathogens. These experimental results suggest that expression of many defense-related genes in compatible host plants might share components of signaling pathways involved in incompatible host-pathogen interactions, but their increased expression has no negative effect on viral infection.     

Expression patterns of defense-related genes in different types of arbuscular mycorrhizal development in wild-type and mycorrhiza-defective mutant tomato

The expression of defense-related genes was analyzed in the interactions of six arbuscular mycorrhizal (AM) fungi with the roots of wild-type tomato (Lycopersicon esculentum Mill.) cv. 76R and of the near-isogenic mycorrhiza-defective mutant rmc. Depending on the fungal species, wild-type tomato forms both major morphological AM types, Arum and Paris. The mutant rmc blocks the penetration of the root surface or invasion of the root cortex by most species of AM fungi, but one fungus has been shown to develop normal mycorrhizas. In the wild-type tomato, accumulation of mRNA representing a number of defense-related genes was low in Arum-type interactions, consistent with findings for this AM morphotype in other plant species. In contrast, Paris-type colonization, particularly by members of the family Gigasporaceae, was accompanied by a substantial transient increase in expression of some defense-related genes. However, the extent of root colonization did not differ significantly in the two wild-type AM morphotypes, suggesting that accumulation of defense gene products per se does not limit mycorrhiza development. In the mutant, interactions in which the fungus failed to penetrate the root lacked significant accumulation of defense gene mRNAs. However, phenotypes in which the fungus penetrated epidermal or hypodermal cells were associated with an enhanced and more prolonged gene expression. These results are discussed in relation to the mechanisms that may underlie the specificity of the interactions between AM fungi and the rmc mutant.     

Global changes in gene expression during compatible and incompatible interactions of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides

ABSTRACT: BACKGROUND: Cowpea, Vigna unguiculata L. Walp., is one of the most important food and forage legumesin the semi-arid tropics. While most domesticated forms of cowpea are susceptible to the rootparasitic weed Striga gesnerioides, several cultivars have been identified that show racespecificresistance. Cowpea cultivar B301 contains the RSG3-301 gene for resistance to S.gesnerioides race SG3, but is susceptible to race SG4z. When challenged by SG3, roots ofcultivar B301 develop a strong resistance response characterized by a hypersensitive reactionand cell death at the site of parasite attachment. In contrast, no visible response occurs inB301 roots parasitized by SG4z. RESULTS: Gene expression in the roots of the cowpea cultivar B301 during compatible (susceptible) andincompatible (resistant) interactions with S. gesnerioides races SG4z and SG3, respectively,were investigated at the early (6 days post-inoculation (dpi)) and late (13 dpi) stages of theresistance response using a Nimblegen custom design cowpea microarray. A total of 111genes were differentially expressed in B301 roots at 6 dpi; this number increased to 2102genes at 13 dpi. At 13 dpi, a total of 1944 genes were differentially expressed duringcompatible (susceptible) interactions of B301 with SG4z . Genes and pathways involved insignal transduction, programmed cell death and apoptosis, and defense response to biotic andbiotic stress were differentially expressed in the early resistance response; at the later timepoint, enrichment was primarily for defense-related gene expression, and genes encodingcomponents of lignifications and secondary wall formation. In compatible interactions (B301- SG4z), multiple defense pathways were repressed, including those involved in ligninbiosynthesis and secondary cell wall modifications, while cellular transport processes fornitrogen and sulfur were increased. CONCLUSION: Distinct changes in global gene expression profiles occur in host roots following successfuland unsuccessful attempted parasitism by Striga. Induction of specific defense related genesand pathways defines components of a unique resistance mechanism. Some genes andpathways up-regulated in the host resistance response to SG3 are repressed in the susceptibleinteractions, suggesting that the parasite is targeting specific components of the host'sdefense. These results add to our understanding of plant-parasite interactions and theevolution of resistance to parasitic weeds.     

The promoter of the Arabidopsis nuclear gene COX5b-1, encoding subunit 5b of the mitochondrial cytochrome c oxidase, directs tissue-specific expression by a combination of positive and negative regulatory elements

In the present work, the promoter of the Arabidopsis thaliana nuclear gene COX5b-1, encoding subunit 5b of the mitochondrial cytochrome c oxidase, has been analysed. For this purpose, plants, stably transformed with different promoter fragments fused to the beta-glucuronidase reporter gene, have been obtained. Histochemical staining indicated that the COX5b-1 promoter directs expression in meristems and in vascular tissues of cotyledons, roots, and hypocotyls, as well as in anthers and pollen and the central leaf vein. Quantitative measurements in extracts prepared from different organs suggested that expression is higher in roots. The analysis of progressive upstream deletions of the promoter suggested the presence of negative regulatory elements, preferentially active in leaves, between nucleotides -609 and -387 from the translation start site. A further deletion down to nucleotide -195 completely abolished expression. The inclusion of sucrose or the cytokinin 6-benzylaminopurine in the culture medium induced COX5b-1 promoter-dependent beta-glucuronidase expression. This induction was observed with all constructs that produced beta-glucuronidase activity. Putative regulatory elements involved in the regulation of other genes were detected in the promoter fragment required for expression. A detailed analysis of these elements will help to elucidate the molecular mechanisms that participate in the expression of this and, possibly, other components of the cytochrome c-dependent respiratory pathway.     

Cloning and characterization of r3b; members of the r3 superfamily of late blight resistance genes show sequence and functional divergence

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.     

Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f. sp. vasinfectum

Microarray analysis of large-scale temporal and tissue-specific plant gene expression changes occurring during a susceptible plant-pathogen interaction revealed different gene expression profile changes in cotton root and hypocotyl tissues. In hypocotyl tissues infected with Fusarium oxysporum f. sp. vasinfectum, increased expression of defense-related genes was observed, whereas few changes in the expression levels of defense-related genes were found in infected root tissues. In infected roots, more plant genes were repressed than were induced, especially at the earlier stages of infection. Although many known cotton defense responses were identified, including induction of pathogenesis-related genes and gossypol biosynthesis genes, potential new defense responses also were identified, such as the biosynthesis of lignans. Many of the stress-related gene responses were common to both tissues. The repression of drought-responsive proteins such as aquaporins in both roots and hypocotyls represents a previously unreported response of a host to pathogen attack that may be specific to vascular wilt diseases. Gene expression results implicated the phytohormones ethylene and auxin in the disease process. Biochemical analysis of hormone level changes supported this observation.     

A common plant cell-wall protein HyPRP1 has dual roles as a positive regulator of cell death and a negative regulator of basal defense against pathogens

Although hybrid proline-rich proteins (HyPRPs) are ubiquitous in plants, little is known about their roles other than as cell-wall structural proteins. We identified the gene HyPRP1 in Capsicum annuum and Nicotiana benthamiana, which encodes a protein containing proline-rich domain and eight-cysteine motif (8CM) that is constitutively expressed in various organs, mostly in the root, but is down-regulated upon inoculation with either incompatible or compatible pathogens. Ectopic expression of HyPRP1 in plants accelerated cell death, showing developmental abnormality with down-regulation of ROS-scavenging genes, and enhanced pathogen susceptibility suppressing expression of defense-related genes. Conversely, silencing of HyPRP1 suppressed pathogen-induced cell death, but enhanced disease resistance, with up-regulation of defense-related genes and inhibition of in planta growth of bacterial pathogens independently of signal molecule-mediated pathways. Furthermore, the secreted 8CM was sufficient for these HyPRP1 functions. Together, our results suggest that a common plant cell-wall structural protein, HyPRP1, performs distinct dual roles in positive regulation of cell death and negative regulation of basal defense against pathogen.     

大豆疫霉菌(Phytophthora sojae)无毒基因Avr1a、Avr1k及Avr3a的分子鉴定

【目的】为大豆疫霉菌(Phytophthora sojae)无毒基因Avr1a、Avr1k和Avr3a快速分子检测提供方法,也为P.sojae其它无毒基因的快速分子检测研究提供依据。【方法】依据GenBank中公布的P.sojae无毒基因Avr1a、Avr1k和Avr3a的序列设计引物,分别筛选出特异性引物,在PCR反应体系和扩增条件优化基础上,对已经接种鉴定过无毒基因Avr1a、Avr1k和Avr3a的86株P.sojae进行PCR检测,建立一套P.sojae无毒基因Avr1a、Avr1k和Avr3a的特异性检测体系。将分子鉴定和接种鉴定结果进行比对,将扩增出的真阳性条带和假阳性条带分别进行胶回收和克隆测序,测序结果分别与3个无毒基因的原序列比对,判定分子标记方法是否适于Avr1a、Avr1k和Avr3a的快速检测。【结果】筛选出的特异性引物均能从含有对应无毒基因的菌株中扩增出约550 bp的条带。Avr1a、Avr1k和Avr3a的分子鉴定及接种鉴定结果符合率依次为45.3%、84.9%和97.7%。3个无毒基因的真阳性条带序列与原序列一致性均达97%以上,Avr1a的假阳性条带与原序列一致性在80%左右,其余2个基因的都在30%以下。【结论】利用Avr1a、Avr1k和Avr3a基因序列分别设计引物建立的检测体系可以用于Avr3a的快速检测,不适于Avr1a的快速检测,是否适合Avr1k的快速检测尚不清楚。     

Expression of a pathogen-induced gene can be mimicked by auxin insensitivity

Following perception of a pathogenic attack, plants are able to develop a strong response with the corresponding activation of a plethora of defense-related genes. In this study we have characterized the mode of expression of the CEVI-1 gene from tomato plants, which encodes an anionic peroxidase. CEVI-1 expression is induced during the course of compatible viral and subviral infections, like many other defense-related genes, but is induced neither in incompatible interactions nor by signal molecules such as salicylic acid, ethylene, or methyl jasmonate. Additionally, CEVI-1 is induced in detached leaf tissues following a pathway distinct from that related to the classical wound response. We also describe the characterization of the structural CEVI-1 gene and compare the mode of expression in different transgenic plant species harboring a CEVI-1::GUS construct. Furthermore, we have isolated mutants in Arabidopsis, called dth mutants, that are deregulated in the control of expression of this gene. From the initial analysis of some of these mutants it seems that activation of CEVI-1 gene expression correlates with a defect in the perception of auxins by the plant. All these results may suggest that, during systemic infections with viruses, auxin homeostasis is one of the components participating in the regulation of the overall defense response.     

Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants

Fumonisin B1 (FB1), a programmed cell death-eliciting toxin produced by the necrotrophic fungal plant pathogen Fusarium moniliforme, was used to simulate pathogen infection in Arabidopsis. Plants infiltrated with 10 microM FB1 and seedlings transferred to agar media containing 1 microM FB1 develop lesions reminiscent of the hypersensitive response, including generation of reactive oxygen intermediates, deposition of phenolic compounds and callose, accumulation of phytoalexin, and expression of pathogenesis-related (PR) genes. Arabidopsis FB1-resistant (fbr) mutants were selected directly by sowing seeds on agar containing 1 microM FB1, on which wild-type seedlings fail to develop. Two mutants chosen for further analyses, fbr1 and fbr2, had altered PR gene expression in response to FB1. fbr1 and fbr2 do not exhibit differential resistance to the avirulent bacterial pathogen Pseudomonas syringae pv maculicola (ES4326) expressing the avirulence gene avrRpt2 but do display enhanced resistance to a virulent isogenic strain that lacks the avirulence gene. Our results demonstrate the utility of FB1 for high-throughput isolation of Arabidopsis defense-related mutants and suggest that pathogen-elicited programmed cell death of host cells may be an important feature of compatible plant-pathogen interactions.     

Genetic and molecular analysis of tomato Cf genes for resistance to Cladosporium fulvum.

In many plant-pathogen interactions resistance to disease is controlled by the interaction of plant-encoded resistance (R) genes and pathogen-encoded avirulence (Avr) genes. The interaction between tomato and the leaf mould pathogen Cladosporium fulvum is an ideal system to study the molecular basis of pathogen perception by plants. A total of four tomato genes for resistance to C. fulvum (Cf-2, Cf-4, Cf-5 and Cf-9) have been isolated from two genetically complex chromosomal loci. Their gene products recognize specific C. fulvum-encoded avirulence gene products (Avr2, Avr4, Avr5 and Avr9) by an unknown molecular mechanism. Cf genes encode extracellular membrane-anchored glycoproteins comprised predominantly of 24 amino acid leucine-rich repeats (LRRs). Cf genes from the same locus encode proteins which are more than 90% identical. Most of the amino-acid sequence differences correspond to the solvent-exposed residues within a beta-strand/beta-turn structural motif which is highly conserved in LRR proteins. Sequence variability within this motif is predicted to affect the specificity of ligand binding. Our analysis of Cf gene loci at the molecular level has shown they comprise tandemly duplicated homologous genes, and suggests a molecular mechanism for the generation of sequence diversity at these loci. Our analysis provides further insight into the molecular basis of pathogen perception by plants and the organization and evolution of R gene loci.