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Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library.

Resistance to grapevine powdery mildew is controlled by Run1, a single dominant gene present in the wild grapevine species, Muscadinia rotundifolia, but absent from the cultivated species, Vitis vinifera. Run1 has been introgressed into V. vinifera using a pseudo-backcross strategy, and genetic markers have previously been identified that are linked to the resistance locus. Here we describe the construction of comprehensive genetic and physical maps spanning the resistance locus that will enable future positional cloning of the resistance gene. Physical mapping was performed using a bacterial artificial chromosome (BAC) library constructed using genomic DNA extracted from a resistant V. vinifera individual carrying Run1 within an introgression. BAC contig assembly has enabled 20 new genetic markers to be identified that are closely linked to Run1, and the position of the resistance locus has been refined, locating the gene between the simple sequence repeat (SSR) marker, VMC4f3.1, and the BAC end sequence-derived marker, CB292.294. This region contains two multigene families of resistance gene analogues (RGA). A comparison of physical and genetic mapping data indicates that recombination is severely repressed in the vicinity of Run1, possibly due to divergent sequence contained within the introgressed fragment from M. rotundifolia that carries the Run1 gene.

Ascomycota↗

Transgenic plants of Vitis vinifera cv. Seyval blanc.

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.

Agrobacterium tumefaciens↗

Cell death and salicylate- and jasmonate-dependent stress responses in Arabidopsis are controlled by single cet genes.

The jasmonic acid (JA)-dependent regulation of the Thi2.1 gene had previously been exploited for setting up a genetic screen for the isolation of signal transduction mutants of Arabidopsis thaliana (L.) Heynh. that constitutively express the thionin gene. Several cet mutants had been isolated which showed a Constitutive Expression of the Thionin gene. These cet mutants, except for one, also showed spontaneous leaf cell necrosis and were up-regulated in the expression of the PR1 gene, reactions often associated with the systemic acquired resistance (SAR) pathway. Four of these cet mutants, cet1, cet2, cet3 and cet4.1 were crossed with the fad triple and coi1 mutants that are blocked at two steps within the JA-dependent signaling pathway, and with transgenic NahG plants that are deficient in salicylic acid (SA) and are unable to activate SAR. Analysis of the various double-mutant lines revealed that the four cet genes act within a signaling cascade at or prior to branch points from which not only JA-dependent signals but also SA-dependent signaling and cell death pathways diverge.

Apoptosis↗

A single recessive mutation in the proteolytic machinery of Arabidopsis chloroplasts impairs photoprotection and photosynthesis upon cold stress.

Intracellular proteases, together with molecular chaperones, are components of the cellular protein quality control system. Although the identity of chloroplast proteases has been revealed in recent years, little is known about their regulation. As a first step towards identifying unknown functional or regulatory components of the chloroplast proteolytic machinery, a genetic screen was devised with the aim of generating Arabidopsis thaliana (L.) Heynh. mutants impaired in chloroplast protein degradation. A streptomycin-resistance gene was fused to a cDNA construct that encodes an unstable mutant of the OE33 protein. This chimeric gene was used for transforming Arabidopsis plants. Analysis of transgenic plants revealed a correlation between streptomycin resistance and accumulation of the recombinant fusion protein. Seeds from transgenic plants that were sensitive to streptomycin were chemically mutagenized and screened for resistance to streptomycin. Such resistance could be due to stabilization of the protein caused by a mutation in the chloroplast proteolytic machinery. Genetic analysis of one of the mutants showed the mutation to be recessive, in a single nuclear gene. Further characterization of the mutant revealed that it was not a result of increased transcription of the transgene. Moreover, chloroplast lysates from the mutant plant showed decreased ATP-dependent degradation of a chloroplast protein substrate, consistent with the conclusion that the mutation impaired the proteolytic machinery. Physiological analysis revealed that upon cold stress, photoprotection and photosynthesis in the mutant was inhibited, suggesting that the chloroplast proteolytic machinery is involved in repairing damage incurred to the photosynthetic machinery upon exposure to cold temperatures.

Acclimatization↗

Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance.

Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.

Ascomycota↗

Transgenic tomato plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases.

Development of effective disease-resistance to a broad-range of pathogens in crops usually requires tremendous resources and effort when traditional breeding approaches are taken. Genetic engineering of disease-resistance in crops has become popular and valuable in terms of cost and efficacy. Due to long-lasting and broad-spectrum of effectiveness against pathogens, employment of systemic acquired resistance (SAR) for the genetic engineering of crop disease-resistance is of particular interest. In this report, we explored the potential of using SAR-related genes for the genetic engineering of enhanced resistance to multiple diseases in tomato. The Arabidopsis NPR1 (nonexpresser of PR genes) gene was introduced into a tomato cultivar, which possesses heat-tolerance and resistance to tomato mosaic virus (ToMV). The transgenic lines expressing NPR1 were normal as regards overall morphology and horticultural traits for at least four generations. Disease screens against eight important tropical diseases revealed that, in addition to the innate ToMV-resistance, the tested transgenic lines conferred significant level of enhanced resistance to bacterial wilt (BW) and Fusarium wilt (FW), and moderate degree of enhanced resistance to gray leaf spot (GLS) and bacterial spot (BS). Transgenic lines that accumulated higher levels of NPR1 proteins exhibited higher levels and a broader spectrum of enhanced resistance to the diseases, and enhanced disease-resistance was stably inherited. The spectrum and degree of these NPR1-transgenic lines are more significant compared to that of transgenic tomatoes reported to date. These transgenic lines may be further explored as future tomato stocks, aiming at building up resistance to a broader spectrum of diseases.

Arabidopsis Proteins↗

Interplay of signaling pathways in plant disease resistance.

Plants are under constant threat of infection by pathogens armed with a diverse array of effector molecules to colonize their host. Plants have, in turn, evolved sophisticated detection and response systems that decipher pathogen signals and induce appropriate defenses. Genetic analysis of plant mutants impaired in mounting a resistance response to invading pathogens has uncovered a number of distinct, but interconnecting, signaling networks that are under both positive and negative control. These pathways operate, at least partly, through the action of small signaling molecules such as salicylate, jasmonate and ethylene. The interplay of signals probably allows the plant to fine-tune defense responses in both local and systemic tissue.

Acetates↗

Priming in plant-pathogen interactions.

Plants can acquire enhanced resistance to pathogens after treatment with necrotizing attackers, nonpathogenic root-colonizing pseudomonads, salicylic acid, beta-aminobutyric acid and many other natural or synthetic compounds. The induced resistance is often associated with an enhanced capacity to mobilize infection-induced cellular defence responses - a process called 'priming'. Although the phenomenon has been known for years, most progress in our understanding of priming has been made only recently. These studies show that priming often depends on the induced disease resistance key regulator NPR1 (also known as NIM1 or SAI1) and that priming has a major effect on the regulation of cellular plant defence responses.

Aminobutyrates↗

Genes controlling expression of defense responses in Arabidopsis--2001 status.

In the past two years, the focus of studies of the genes controlling expression of defense responses in Arabidopsis has shifted from the identification of mutants to gene isolation and the ordering of genes within branches of the signal transduction networks. It is now clear that gene-for-gene resistance can be mediated through at least three genetically distinguishable pathways. Additional genes affecting salicylic-acid-dependent signaling have been identified, and double-mutant analyses have begun to reveal the order in which they act. Genes required for jasmonic-acid-dependent signaling and for induced systemic resistance have also been identified.

Antifungal Agents↗

Mechanisms of plant resistance to viruses.

Plants have evolved in an environment rich with microorganisms that are eager to capitalize on the plants' biosynthetic and energy-producing capabilities. There are approximately 450 species of plant-pathogenic viruses, which cause a range of diseases. However, plants have not been passive in the face of these assaults, but have developed elaborate and effective defence mechanisms to prevent, or limit, damage owing to viral infection. Plant resistance genes confer resistance to various pathogens, including viruses. The defence response that is initiated after detection of a specific virus is stereotypical, and the cellular and physiological features associated with it have been well characterized. Recently, RNA silencing has gained prominence as an important cellular pathway for defence against foreign nucleic acids, including viruses. These pathways function in concert to result in effective protection against virus infection in plants.

Genes, Plant↗

The tomato Rme1 locus is required for Mi-1-mediated resistance to root-knot nematodes and the potato aphid.

The tomato Mi-1 gene confers resistance against root-knot nematodes (Meloidogyne spp.) and a biotype of the potato aphid (Macrosiphum euphorbiae). Four mutagenized Mi-1/Mi-1 tomato populations were generated and screened for altered root-knot nematode resistance. Four independent mutants belonging to two phenotypic classes were isolated. One mutant was chosen for further analyzes; rme1 (for resistance to Meloidogyne) exhibited levels of infection comparable with those found on susceptible controls. Molecular and genetic data confirmed that rme1 has a single recessive mutation in a locus different from Mi-1. Cross-sections through galls formed by feeding nematodes on rme1 roots were identical to sections from galls of susceptible tomato roots. In addition to nematode susceptibility, infestation of rme1 plants with the potato aphid showed that this mutation also abolished aphid resistance. To determine whether Rme1 functions in a general disease-resistance pathway, the response against Fusarium oxysporum f.sp. lycopersici race 2, mediated by the I-2 resistance gene, was studied. Both rme1 and the wild type plants were equally resistant to the fungal pathogen. These results indicate that Rme1 does not play a general role in disease resistance but may be specific for Mi-1-mediated resistance.

Animals↗

Characterization of Arabidopsis enhanced disease susceptibility mutants that are affected in systemically induced resistance.

In Arabidopsis, the rhizobacterial strain Pseudomonas fluorescens WCS417r triggers jasmonate (JA)- and ethylene (ET)-dependent induced systemic resistance (ISR) that is effective against different pathogens. Arabidopsis genotypes unable to express rhizobacteria-mediated ISR against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) exhibit enhanced disease susceptibility towards this pathogen. To identify novel components controlling induced resistance, we tested 11 Arabidopsis mutants with enhanced disease susceptibility (eds) to pathogenic P. syringae bacteria for WCS417r-mediated ISR and pathogen-induced systemic acquired resistance (SAR). Mutants eds4-1, eds8-1 and eds10-1 failed to develop WCS417r-mediated ISR, while mutants eds5-1 and eds12-1 failed to express pathogen-induced SAR. Whereas eds5-1 is known to be blocked in salicylic acid (SA) biosynthesis, analysis of eds12-1 revealed that its impaired SAR response is caused by reduced sensitivity to this molecule. Analysis of the ISR-impaired eds mutants revealed that they are non-responsive to induction of resistance by methyl jasmonate (MeJA) (eds4-1, eds8-1 and eds10-1), or the ET precursor 1-aminocyclopropane-1-carboxylate (ACC) (eds4-1 and eds10-1). Moreover, eds4-1 and eds8-1 showed reduced expression of the plant defensin gene PDF1.2 after MeJA and ACC treatment, which was associated with reduced sensitivity to either ET (eds4-1) or MeJA (eds8-1). Although blocked in WCS417r-, MeJA- and ACC-induced ISR, eds10-1 behaved normally for several other responses to MeJA or ACC. The results indicate that EDS12 is required for SAR and acts downstream of SA, whereas EDS4, EDS8 and EDS10 are required for ISR acting either in JA signalling (EDS8), ET signalling (EDS4), or downstream JA and ET signalling (EDS10) in the ISR pathway.

Acetates↗

The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease.

We describe the characterization of a novel gain-of-function Arabidopsis mutant, dll1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection. Despite the constitutive expression of defense-related responses, dll1 is unable to suppress the growth of virulent pathogens. However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses. The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants. This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease. Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death. Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process.

Apoptosis↗

Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance.

The N gene, a member of the Toll-IL-1 homology region-nucleotide binding site-leucine-rich repeat region (LRR) class of plant resistance genes, encodes two transcripts, N(S) and N(L), via alternative splicing of the alternative exon present in the intron III. The N(S) transcript, predicted to encode the full-length N protein containing the Toll-IL-1 homology region, nucleotide binding site, and LRR, is more prevalent before and for 3 hr after tobacco mosaic virus (TMV) infection. The N(L) transcript, predicted to encode a truncated N protein (N(tr)) lacking 13 of the 14 repeats of the LRR, is more prevalent 4-8 hr after TMV infection. Plants harboring a cDNA-N(S) transgene, capable of encoding an N protein but not an N(tr) protein, fail to exhibit complete resistance to TMV. Transgenic plants containing a cDNA-N(S)-bearing intron III and containing 3' N-genomic sequences, encoding both N(S) and N(L) transcripts, exhibit complete resistance to TMV. These results suggest that both N transcripts and presumably their encoded protein products are necessary to confer complete resistance to TMV.

Alternative Splicing↗