Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.

The role of RNA silencing as an antiviral defense mechanism in fungi was examined by testing the effect of dicer gene disruptions on mycovirus infection of the chestnut blight fungus Cryphonectria parasitica. C. parasitica dicer-like genes dcl-1 and dcl-2 were cloned and shown to share a high level of predicted amino acid sequence identity with the corresponding dicer-like genes from Neurospora crassa [Ncdcl-1 (50.5%); Ncdcl-2 (38.0%)] and Magnaporthe oryzae [MDL-1 (45.6%); MDL-2 (38.0%)], respectively. Disruption of dcl-1 and dcl-2 resulted in no observable phenotypic changes relative to wild-type C. parasitica. Infection of ⌬dcl-1 strains with hypovirus CHV1-EP713 or reovirus MyRV1-Cp9B21 resulted in phenotypic changes that were indistinguishable from that exhibited by wildtype strain C. parasitica EP155 infected with these same viruses. In stark contrast, the ⌬dcl-2 and ⌬dcl-1/⌬dcl-2 mutant strains were highly susceptible to mycovirus infection, with CHV1-EP713-infected mutant strains becoming severely debilitated. Increased viral RNA levels were observed in the ⌬dcl-2 mutant strains for a hypovirus CHV1-EP713 mutant lacking the suppressor of RNA silencing p29 and for wild-type reovirus MyRV1-Cp9B21. Complementation of the ⌬dcl-2 strain with the wild-type dcl-2 gene resulted in reversion to the wild-type response to virus infection. These results provide direct evidence that a fungal dicer-like gene functions to regulate virus infection.Cryphonectria parasitica ͉ Dicer ͉ hypovirus ͉ mycoreovirus ͉ double-stranded RNA R NA-mediated, sequence-specific suppression of gene expression, termed RNA silencing, has been described as posttranscriptional gene silencing in plants (1, 2), RNA interference (RNAi) in animals (3), and quelling in fungi (4). A common feature of RNA silencing in these different organisms is the processing of structured or dsRNA into small interfering RNAs (siRNAs) of 21-24 nt by RNase III-like endonucleases termed Dicers. These siRNAs then are incorporated into an RNAinduced silencing complex that guides sequence-specific degradation or translational repression of homologous RNA in the cytoplasm or DNA or histone methylation of target sequences in the nucleus (reviewed in refs. 5 and 6).In plants and animals, the RNA silencing pathway also produces microRNAs (miRNAs) from genome-encoded RNA hairpins that are involved in developmental regulation (reviewed in refs. 7 and 8). miRNAs have not been identified in fungal genomes (9, 10). Thus, RNA silencing in fungi generally is thought to serve primarily as a defense mechanism against invasive nucleic acids and viruses (10). RNA silencing plays a key antiviral defense role in plants (reviewed in refs. 11 and 12) and recently has been demonstrated to influence virus replication in animal cells (reviewed in ref. 13). Although silencing of transposons has been reported in fungi (14), there currently are no reports of RNA silencing functioning as a fungal antiviral defense mechanism.Mechanisms underlying RNA silencing in fungi have been elucidated prim...

Dicer gene dcl2, required for the RNA silencing antiviral defense response in the chestnut blight fungus Cryphonectria parasitica, is inducible upon mycovirus infection and promotes viral RNA recombination. We now report that the antiviral defense response requires only one of the four C. parasitica Argonaute-like protein genes, agl2. The agl2 gene is required for the virus-induced increase in dcl2 transcript accumulation. Agl2 and dcl2 transcripts accumulated to much higher levels in response to hairpin RNA production or infection by a mutant CHV1-EP713 hypovirus lacking the suppressor of RNA silencing p29 than to wild-type CHV1-EP713. Similar results were obtained for an agl2-promoter/EGFP-reporter construct, indicating that p29-mediated repression of agl2 transcript accumulation is promoter-dependent. Significantly, the agl2 deletion mutant exhibited stable maintenance of non-viral sequences in recombinant hypovirus RNA virus vectors and the absence of hypovirus-defective interfering (DI) RNA production. These results establish a key role for an Argonaute gene in the induction of an RNA silencing antiviral defense response and the promotion of viral RNA recombination. They also provide evidence for a mechanism by which a virus-encoded RNA silencing suppressor represses the transcriptional induction of an RNA silencing component.Cryphonectria parasitica ͉ mycovirus ͉ defective interfering RNA ͉ silencing suppressor ͉ RNA virus vector A n RNA-based antiviral defense response, related to RNA interference (1), serves as a key component of the innate immunity repertoire in plants, invertebrates, and fungi (2, 3). Common elements of this response across Kingdoms include the action of conserved ribonucleases: members of the Dicer-like and Argonaute-like protein families (4). Dicer nucleases recognize viral double-stranded (ds) and structured RNAs and use the associated RNase III-type activity to process these RNAs into small RNAs of 21-24 nts in length, termed virus-derived small (vs) RNAs. The vsRNAs are incorporated into an effector complex with the aid of an Argonaute family protein. One strand of the vsRNA is removed and the remaining guide strand then targets the effector complex to the cognate viral RNA, which is cleaved, or sliced, by the Argonaute-associated RNase H-like activity.Four Dicer proteins drive the RNA silencing pathways in the model plant, Arabidopsis thaliana (5). All four Dicers have been implicated in antiviral RNA silencing (3, 6-8). Studies with Drosophila melanogaster (9, 10) and with mosquitoes (11) have identified a role for Dicer-2, the Dicer involved in siRNA production, in insect antiviral defense. The contribution to antiviral defense of the second Dicer, Dicer-1, remains unclear due to its essential role in microRNA processing and development (12).Antiviral RNA silencing has been demonstrated in the filamentous Ascomycete fungi Cryphonectria parasitica, the chestnut blight fungus (13), and Aspergillus nidulans (14). Only one of the two C. parasitica Dicer genes, dcl2, was shown to be...