Dodder infection induces the expression of a pathogenesis-related gene of the family PR-10 in alfalfa

A full-length cDNA, PPRG2, representing a gene highly expressed in dodder (Cuscuta trifolii Bab et. Gibs)-infected alfalfa (Medicago sativa L.) stems was isolated by differential screening. The predicted protein contains 157 amino acids and belongs to the PR-10 family of the pathogenesis-related genes with putative ribonuclease activities. Northern hybridizations showed that PPRG2 is transcribed in root and crops of uninfected alfalfa and is induced not only by dodder attack but also by bacterial infections and a large variety of environmental stresses.     

Functional analysis of Ras in Colletotrichum trifolii

Ras is a small monomeric GTP binding protein that transduces signals for growth and differentiation of eukaryotic organisms. Previously, a unique ras gene, designated Ct-ras, was cloned from the alfalfa fungal phytopathogen, Colletotrichum trifolii. Expression of Ct-Ras in mouse fibroblast cells (NIH3T3) demonstrated that Ct-ras is functionally similar to the mammalian ras genes since activating mutations of Ct-ras caused oncogenic phenotypes in nu/nu mice, including tumors. In C. trifolii, activated 'oncogenic' Ras (Val2) induced abnormal hyphal proliferation, defects in polarized growth and significantly reduced differentiation such as conidiation and appressorium formation in a nutrient dependent manner. Gene disruption of ct-ras was lethal. To further evaluate the function of Ct-Ras in C. trifolii, three different approaches were used: overexpression of cytosolic Ras by CAAX box deletion; expression of dominant negative Ct-RasT22N; and antisense ct-ras expression. Results showed that suppression of Ct-Ras activity significantly decreases fungal germination frequencies and hyphal growth rates. Taken together, these data suggest involvement of Ct-Ras in regulation of fungal cell growth and differentiation.     

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.     

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.     

Colletotrichum trifolii mutants disrupted in the catalytic subunit of cAMP-dependent protein kinase are nonpathogenic

Colletotrichum trifolii is the fungal pathogen of alfalfa that causes anthracnose disease. For successful plant infection, this fungus must undergo a series of morphological transitions following conidial attachment, including germination and subsequent differentiation, resulting in appressorium formation. Our previous studies with pharmacological effectors of signaling pathways have suggested the involvement of cyclic AMP (cAMP)-dependent protein kinase (PKA) during these processes. To more precisely evaluate the role of PKA in C. trifolii morphogenesis, the gene encoding the catalytic (C) subunit of PKA (Ct-PKAC) was isolated, sequenced, and inactivated by gene replacement. Southern blot analysis with C. trifolii genomic DNA suggested that Ct-PKAC is a single-copy gene. Northern (RNA) blot analysis with total RNA from different fungal growth stages indicated that the expression of this gene was developmentally regulated. When Ct-PKAC was insertionally inactivated by gene replacement, the transformants showed a small reduction in growth relative to the wild type and conidiation patterns were altered. Importantly, PKA-deficient strains were unable to infect intact alfalfa (host) plants, though only a slight delay was observed in the timing for conidial germination and appressorial formation in the Ct-PKAC disruption mutants. Moreover, these mutants were able to colonize host tissues following artificial wounding, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggest that the defect in pathogenicity is likely due to a failure in penetration. Our results demonstrate that PKA has an important role in regulating the transition between vegetative growth and conidiation, and is essential for pathogenic development in C. trifolii.     

Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.     

The plant cell defense and Agrobacterium tumefaciens

We previously identified changes in gene expression in Ageratum conyzoides plant cells inoculated with Agrobacterium tumefaciens by using cDNA-AFLP. Here, we show that a subset of defense-related genes is differentially regulated by an Agrobacterium attachment-deficient mutant. The expression pattern triggered by this mutant is similar to that induced by inoculation with non-pathogenic bacteria. We also observed that the expression level of the defense genes was inversely correlated with the efficiency of transformation by Agrobacterium. We propose that the plant defense system has an important role in controlling infection and transformation and that Agrobacterium may dampen some plant defense responses.     

Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.     

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.     

Regeneration of foreign genes co-transformed plants of Medicago sativa L by Agrobacterium rhizogenes

Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of transgenic plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.     

Independent action and contrasting phenotypes of resistance genes against spotted alfalfa aphid and bluegreen aphid in Medicago truncatula

Host resistance to aphids is poorly understood. Medicago truncatula, a model legume and cultivated pasture species, was used to elucidate defense against two aphid species, Therioaphis trifolii f. maculata (spotted alfalfa aphid, SAA) and Acyrthosiphon kondoi (bluegreen aphid, BGA). Aphid performance and plant damage were compared between near-isogenic cultivars, Mogul and Borung, that differ in resistance to both aphids. Analyses of aphid resistance in Mogul x Borung F2 plants and their progeny revealed modes of action and chromosome locations of resistance genes. Separate genes were identified for SAA resistance (TTR) and BGA resistance (AKR); both mapped to chromosome 3 but were found to act independently to reduce survival and growth of their target aphid species. The TTR locus controls distinct, and contrasting, local and systemic plant responses between the near-isogenic cultivars. TTR-mediated plant responses imply interaction between a resistance factor(s) in vascular tissue and a bioactive component(s) of SAA saliva. Features of both resistance traits suggest homology to aphid resistance in other legumes; elucidation of their molecular mechanisms will likely apply to other aphid-plant interactions.     

水稻抗稻瘟病基因资源与分子育种策略

稻瘟病是水稻主要病害之一。利用抗病品种是防治稻瘟病最经济、有效和安全的措施。近年来,随着植物先天免疫机制、抗病分子生物学以及水稻和稻瘟菌基因组学研究的不断深入,一系列参与病原菌识别和防卫信号传导与应答,以及外源抗菌蛋白、病原菌激发子等抗病相关基因陆续被鉴定和克隆,为提高水稻抗稻瘟病能力提供了一些新的基因资源、育种策略和技术。本文概述了国内外近年来克隆的主要抗稻瘟病基因资源及其在分子育种研究中应用的进展,提出了通过转基因手段整合不同防卫反应关键调控基因的抗稻瘟病聚合育种策略。     

Regeneration of foreign genes co-transformed plants ofMedicago sativa L byAgrobacterium rhizogenes

Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of trans-genie plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.