No abstract

Analysis of viral and bacterial pathogenesis has revealed common themes in the ways in which plants and animals respond to pathogenic agents. Pathogenic bacteria use macromolecule delivery systems (types III and IV) to deliver microbial avirulence proteins and transfer DNA-protein complexes directly into plant cells. The molecular events that constitute critical steps of plant-pathogen interactions seem to involve ligand-receptor mechanisms for pathogen recognition and the induction of signal transduction pathways in the plant that lead to defense responses. Unraveling the molecular basis of disease resistance pathways has laid a foundation for the rational design of crop protection strategies.

Plant genomes contain large numbers of cell surface leucine-rich repeat (LRR) and intracellular nucleotide binding (NB)-LRR immune receptors encoded by resistance (R) genes that recognize specific pathogen effectors and trigger resistance responses. The unregulated expression of NB-LRR genes can trigger autoimmunity in the absence of pathogen infection and inhibit plant growth. Despite the potential serious consequence on agricultural production, the mechanisms regulating R-gene expression are not well understood. We identified microRNA (miRNA) progenitor genes precursor transcripts, and two miRNAs [nta-miR6019 (22-nt) and ntamiR6020 (21-nt)] that guide cleavage of transcripts of the Toll and Interleukin-1 receptor-NB-LRR immune receptor N from tobacco that confers resistance to tobacco mosaic virus (TMV). We further showed that cleavage by nta-miR6019 triggers RNA-dependent RNA polymerase 6-and ribonuclease Dicer-like 4-dependent biogenesis of 21-nt secondary siRNAs "in phase" with the 22-nt miR6019 cleavage site. Furthermore, we found that processing of the 22-nt nta-miR6019 depended on an asymmetric bulge caused by mismatch in the nta-miR6019 precursor. Interestingly, coexpression of N with nta-miR6019 and nta-miR6020 resulted in attenuation of N-mediated resistance to TMV, indicating that these miRNAs have functional roles in NB-LRR regulation. Using a bioinformatics approach, we identified six additional 22-nt miRNA and two 21-nt miRNA families from three Solanaceae species-tobacco, tomato, and potato. We show that members of these miRNA families cleave transcripts of predicted functional R genes and trigger production of phased secondary 21-nt siRNAs. Our results demonstrate a conserved role for miRNAs and secondary siRNAs in NB-LRR/LRR immune receptor gene regulation and pathogen resistance in Solanaceae.

Plant breeders have used disease resistance genes ( R genes) to control plant disease since the turn of the century. Molecular cloning of R genes that enable plants to resist a diverse range of pathogens has revealed that the proteins encoded by these genes have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms for the expression of resistance to a wide range of unrelated pathogens. Characterization of the molecular signals involved in pathogen recognition and of the molecular events that specify the expression of resistance may lead to novel strategies for plant disease control.

SummaryComparative genomics provides a tool to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. In this report, we adopted a local RGA approach using genomic information from the model Solanaceous plant tomato to isolate R3a, a potato gene that confers race-specific resistance to the late blight pathogen Phytophthora infestans. R3a is a member of the R3 complex locus on chromosome 11. Comparative analyses of the R3 complex locus with the corresponding I2 complex locus in tomato suggest that this is an ancient locus involved in plant innate immunity against oomycete and fungal pathogens. However, the R3 complex locus has evolved after divergence from tomato and the locus has experienced a significant expansion in potato without disruption of the flanking colinearity. This expansion has resulted in an increase in the number of R genes and in functional diversification, which has probably been driven by the co-evolutionary history between P. infestans and its host potato. Constitutive expression was observed for the R3a gene, as well as some of its paralogues whose functions remain unknown.

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 NL, via alternative splicing of the alternative exon present in the intron III. The NS 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 NL 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-NS 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-NS-bearing intron III and containing 3 Ngenomic sequences, encoding both N S and NL 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.

We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.

The tobacco N gene is a member of the Toll-interleukin-1 receptor͞nucleotide-binding site͞leucine-rich repeat (TIR-NBS-LRR) class of plant resistance (R) genes and confers resistance to tobacco mosaic virus (TMV). We investigated the importance of specific domains of N in inducing TMV resistance, by examining various N deletion and point mutations that introduce single amino acid substitution mutants in vivo. Our deletion analysis suggests that the TIR, NBS, and LRR domains play an indispensable role in the induction of resistance responses against TMV. We show that amino acids conserved among the Toll͞IL-1R͞plant R gene TIR domain and NBS-containing proteins play a critical role in Nmediated TMV resistance. Some loss-of-function N alleles such as the TIR deletion and point mutations in the NBS (G216A͞E͞V͞R, G218R, G219D, K222E͞N, and T223A͞N) interfere with the wildtype N function and behave like dominant negative mutations. These F1 plants mount a hypersensitive response (HR) that is indistinguishable from that of the wild-type N plants, yet TMV was able to move systemically, causing a systemic hypersensitive response (SHR). Many amino acid substitutions in the TIR, NBS, and LRR domains of N lead to a partial loss-of-function phenotype. These mutant plants mount delayed HR compared with the wildtype N plants and fail to contain the virus to the infection site. In addition, some partial loss-of-function alleles (W82S͞A, W141S͞A, G218V͞S, and G219V) interfere with the wild-type N function, leading to SHR. The partial loss-of-function and dominant negative mutant alleles described in this report will be useful in furthering our understanding of the TIR-NBS-LRR class of R genes. I n plants, R genes are hypothesized to encode receptors that interact directly or indirectly with ligands produced by the corresponding invading pathogen avirulence (Avr) genes. This initial recognition event triggers defense responses that halt pathogen spread. Absence of an R gene in the plant or specific Avr gene in the invading pathogen results in successful colonization of the pathogen on the plant and further development of disease. This R-Avr-mediated defense response in plants is termed a ''gene-for-gene'' type of resistance (1).The typical plant defense response includes the hypersensitive response (HR) or localized cell death at the site of pathogen ingress and containment of the pathogen to the infection site. The HR shares many characteristic features with nematode and animal-programmed cell apoptosis and may play a direct role in the restriction of a pathogen to the infection site (2, 3). A plethora of biochemical and physiological changes coincides with R gene-triggered resistance and host cell death (4). Some of the processes that are associated with gene-for-gene resistance are generation of reactive oxygen species and nitric oxide, production of antimicrobial compounds, lipid peroxidation, ion fluxes, cell wall strengthening, lignin deposition, and induction of defense genes. The local induced HR response often correlates...