Background: RNA-seq and microarray are the two popular methods employed for genome-wide transcriptome profiling. Current comparison studies have shown that transcriptome quantified by these two methods correlated well. However, none of them have addressed if they complement each other, considering the strengths and the limitations inherent with them. The pivotal requirement to address this question is the knowledge of a well known data set. In this regard, HrpX regulome from pathogenic bacteria serves as an ideal choice as the target genes of HrpX transcription factor are well studied due to their central role in pathogenicity.Results: We compared the performance of RNA-seq and microarray in their ability to detect known HrpX target genes by profiling the transcriptome from the wild-type and thehrpXmutant strains of γ-ProteobacteriumXanthomonas citrisubsp.citri. Our comparative analysis indicated that gene expression levels quantified by RNA-seq and microarray well-correlated both at absolute as well as relative levels (Spearman correlation-coefficient, r_s> 0.76). Further, the expression levels quantified by RNA-seq and microarray for the significantly differentially expressed genes (DEGs) also well-correlated with qRT-PCR based quantification (r_s = 0.58 to 0.94). Finally, in addition to the 55 newly identified DEGs, 72% of the already known HrpX target genes were detected by both RNA-seq and microarray, while, the remaining 28% could only be detected by either one of the methods.Conclusions: This study has significantly advanced our understanding of the regulome of the critical transcriptional factor HrpX. RNA-seq and microarray together provide a more comprehensive picture of HrpX regulome by uniquely identifying new DEGs. Our study demonstrated that RNA-seq and microarray complement each other in transcriptome profiling.

Summary: Huanglongbing ( HLB ) or citrus greening disease is a destructive disease of citrus worldwide, which is associated with C andidatus L iberibacter asiaticus. This phloem‐limited fastidious pathogen is transmitted by the A sian citrus psyllid, D iaphorina citri , and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that, during infection, C a . L . asiaticus differentially expresses the genes critical for its survival and/or pathogenicity in either host. To test this hypothesis, quantitative reverse transcription‐polymerase chain reaction was performed to compare the gene expression of C a . L . asiaticus in planta and in psyllid. Overall, 381 genes were analysed for their gene expression in planta and in psyllid. Among them, 182 genes were up‐regulated in planta compared with in psyllid ( P < 0.05), 16 genes were up‐regulated in psyllid ( P < 0.05) and 183 genes showed no statistically significant difference ( P ≥ 0.05) in expression between in planta and in psyllid. Our study indicates that the expression of the C a . L . asiaticus genes involved in transcriptional regulation, transport system, secretion system, flagella assembly, metabolic pathway and stress resistance are changed significantly in a host‐specific manner to adapt to the distinct environments of plant and insect. To our knowledge, this is the first large‐scale study to evaluate the differential expression of C a . L . asiaticus genes in a plant host and its insect vector.

Citrus canker is caused by Xanthomonas citri subsp. citri and is one of the most devastating diseases on citrus plants. To investigate the virulence mechanism of this pathogen, a mutant library of strain 306 containing approximately 22,000 mutants was screened for virulence-deficient mutants in grapefruit (Citrus paradise). Eighty-two genes were identified that contribute to citrus canker symptom development caused by X. citri subsp. citri. Among the 82 identified genes, 23 genes were classified as essential genes, as mutation of these genes caused severe reduction of bacterial growth in M9 medium. The remaining 59 genes were classified as putative virulence-related genes that include 32 previously reported virulence-related genes and 27 novel genes. The 32 known virulence-related genes include genes that are involved in the type III secretion system (T3SS) and T3SS effectors, the quorum-sensing system, extracellular polysaccharide and lipopolysaccharide synthesis, and general metabolic pathways. The contribution to pathogenesis by nine genes (pthA4, trpG, trpC, purD, hrpM, peh-1, XAC1230, XAC1548, and XAC3049) was confirmed by complementation assays. We further validated the mutated genes and their phenotypes by analyzing the EZ-Tn5 insertion copy number using Southern blot analysis. In conclusion, we have significantly advanced our understanding of the putative genetic determinants of the virulence mechanism of X. citri subsp. citri by identifying 59 putative virulence-related genes, including 27 novel genes.

Bacterial citrus canker disease, which is caused by Xanthomonas citri subsp. citri , is one of the most devastating diseases of citrus plants. In this study, we characterized the role of the two-component regulatory system ColR/ColS in the pathogenicity of X. citri subsp. citri. colS mutants (256A10 and 421E7), colR mutants (386C6 and 417E10), and a colR colS double mutant (306DSR) all lost pathogenicity and produced no symptoms on grapefruit leaves inoculated by either pressure infiltration or the spray method. The pathogenicity defect of the colS , colR , and colR colS mutants could be complemented using the wild-type colS , colR , and colR colS genes, respectively. Mutation of colS or colR significantly reduced X. citri subsp. citri growth in planta . The ColR/ColS system also played important roles in bacterial biofilm formation in glass tubes and on leaf surfaces, lipopolysaccharide (LPS) production, catalase activity, and tolerance of environmental stress, including phenol, copper, and hydrogen peroxide. Furthermore, quantitative reverse transcription-PCR assays demonstrated that the ColR/ColS system positively regulated the expression of important virulence genes, including hrpD 6, hpaF , the O-antigen LPS synthesis gene rfbC , and the catalase gene katE . Overall, our data indicate that the two-component regulatory system ColR/ColS is critical for X. citri subsp. citri virulence, growth in planta , biofilm formation, catalase activity, LPS production, and resistance to environmental stress.

Metabolic co-regulation between biosynthetic pathways for secondary metabolites is common in microbes and can play an important role in microbial interactions. Here, we describe a novel mechanism of metabolic co-regulation in which an intermediate in one pathway is converted into signals that activate a second pathway. Our study focused on the co-regulation of 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin, two antimicrobial metabolites produced by the soil bacterium Pseudomonas protegens. We show that an intermediate in DAPG biosynthesis, phloroglucinol, is transformed by a halogenase encoded in the pyoluteorin gene cluster into mono- and di-chlorinated phloroglucinols. The chlorinated phloroglucinols function as intra- and inter-cellular signals that induce the expression of pyoluteorin biosynthetic genes, pyoluteorin production, and pyoluteorin-mediated inhibition of the plant-pathogenic bacterium Erwinia amylovora. This metabolic co-regulation provides a strategy for P. protegens to optimize the deployment of secondary metabolites with distinct roles in cooperative and competitive microbial interactions.