Concomitant Regulation by a LacI-Type Transcriptional Repressor XylR on Genes Involved in Xylan and Xylose Metabolism and the Type III Secretion System in Rice Pathogen Xanthomonas oryzae pv. oryzae

The hypersensitive response and pathogenicity (hrp) genes of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, encode components of the type III secretion system and are essential for virulence. Expression of hrp genes is regulated by two key hrp regulators, HrpG and HrpX; HrpG regulates hrpX and hrpA, and HrpX regulates the other hrp genes on hrpB-hrpF operons. We previously reported the sugar-dependent quantitative regulation of HrpX; the regulator highly accumulates in the presence of xylose, followed by high hrp gene expression. Here, we found that, in a mutant lacking the LacI-type transcriptional regulator XylR, HrpX accumulation and hrp gene expression were high even in the medium without xylose, reaching the similar levels present in the wild type incubated in the xylose-containing medium. XylR also negatively regulated one of two xylose isomerase genes (xylA2 but not xylA1) by binding to the motif sequence in the upstream region of the gene. Xylose isomerase is an essential enzyme in xylose metabolism and interconverts between xylose and xylulose. Our results suggest that, in the presence of xylose, inactivation of XylR leads to greater xylan and xylose utilization and, simultaneously, to higher accumulation of HrpX, followed by higher hrp gene expression in the bacterium.

Evidence for a Novel Phylotype of Pseudomonas syringae Causing Bacterial Leaf Blight of Cantaloupe in China

Bacterial leaf blight (BLB) has caused severe yield losses in cantaloupe (Cucumis melo L.) in the major melon-growing regions of China since the beginning of the twentieth century. Historically, Pseudomonas syringae pv. lachrymans was considered to be the causal agent of BLB of cantaloupe and angular leaf spot of cucumber. In the process of characterizing bacteria isolated from cantaloupe, we observed that putative P. syringae pv. lachrymans yielded negative results in P. syringae pv. lachrymans-specific PCR assays. This suggested that the P. syringae pv. lachrymans-like strains from cantaloupe were distinct from those recovered from cucumber. To investigate the differences between P. syringae pv. lachrymans-like strains isolated from cantaloupe and cucumber, 13 P. syringae strains isolated from cantaloupe [12 from China and 1 from Zimbabwe (NCPPB2916)] and 7 additional P. syringae reference strains were analyzed by catabolic profiling, phylogenetic analysis by multilocus sequence analysis (MLSA) and pathogenicity tests on cantaloupe leaflets. Catabolic profiling and MLSA based on 10 housekeeping genes and 2 hypersensitive response and pathogenicity (hrp) genes allowed us to differentiate strains isolated from cantaloupe and cucumber. Pseudomonas syringae pv. lachrymans strains isolated from cucumber clustered with genomospecies 2, and 13 P. syringae strains isolated from cantaloupe belonged to genomospecies 1. While all cantaloupe strains were closely related to P. syringae pv. aptata, they could be differentiated from this pathovar based on metabolic tests and MLSA. Pathogenicity tests showed that all strains isolated from cantaloupe and cucumber were only pathogenic on their original hosts. Based on these observations we conclude that P. syringae pv. lachrymans strains recovered from cantaloupe in China represent a novel phylotype.

Systematic Mutagenesis of All Predicted gntR Genes in Xanthomonas campestris pv. campestris Reveals a GntR Family Transcriptional Regulator Controlling Hypersensitive Response and Virulence

The GntR family is one of the most abundant and widely distributed groups of helix-turn-helix transcriptional regulators in bacteria. Six open reading frames in the genome of the plant pathogen Xanthomonas campestris pv. campestris were predicted to encode GntR regulators. All six of the predicted GntR-encoding genes were individually mutagenized and mutants from five of them were successfully obtained. Plant disease response assays revealed that one, whose product belongs to the YtrA subfamily and has been named HpaR1, is involved in the hypersensitive response (HR) and virulence. Electrophoretic mobility shift assays and in vitro transcription assays revealed that HpaR1 could repress its own transcription level through binding to its promoter sequence, indicating an autoregulatory feedback inhibition mechanism for HpaR1 expression. Promoter-gusA reporter and reverse-transcription polymerase chain reaction analyses revealed that HpaR1 positively and negatively affects the expression of HR and pathogenicity (hrp) genes in host plant and standard media, respectively. Constitutive expression of the key hrp regulator, hrpG, in the hpaR1 mutant could bypass the requirement of HpaR1 for the induction of wild-type HR, suggesting that HpaR1 regulates the expression of hrp genes that encode the type III secretion system via hrpG.

A Novel Regulatory Role of HrpD6 in Regulating hrp-hrc-hpa Genes in Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak in the model plant rice, possesses a hypersensitive response and pathogenicity (hrp), hrp-conserved (hrc), hrp-associated (hpa) cluster (hrp-hrc-hpa) that encodes a type III secretion system (T3SS) through which T3SS effectors are injected into host cells to cause disease or trigger plant defenses. Mutations in this cluster usually abolish the bacterial ability to cause hypersensitive response in nonhost tobacco and pathogenicity in host rice. In Xanthomonas spp., these genes are generally assumed to be regulated by the key master regulators HrpG and HrpX. However, we present evidence that, apart from HrpG and HrpX, HrpD6 is also involved in regulating the expression of hrp genes. Interestingly, the expression of hpa2, hpa1, hpaB, hrcC, and hrcT is positively controlled by HrpD6. Transcriptional expression assays demonstrated that the expression of the hrcC, hrpD5, hrpE, and hpa3 genes was not completely abolished by hrpG and hrpX mutations. As observed in analysis of their corresponding mutants, HrpG and HrpX exhibit contrasting gene regulation, particularly for hpa2 and hrcT. Other two-component system regulators (Zur, LrpX, ColR/S, and Trh) did not completely inhibit the expression of hrcC, hrpD5, hrpE, and hpa3. Immunoblotting assays showed that the secretion of HrpF, which is an HpaB-independent translocator, is not affected by the mutation in hrpD6. However, the mutation in hrpD6 affects the secretion of an HpaB-dependent TAL effector, AvrXa27. These novel findings suggest that, apart from HrpG and HrpX, HrpD6 plays important roles not only in the regulation of hrp genes but also in the secretion of TAL effectors.