Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

Background

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood.

Results

We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R.

Conclusion

About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.     

Virulence analysis and genetic diversity of Xanthomonas campestris pv. campestris causing black rot of crucifers

Xanthomonas campestris pv. campestris (Xcc) is a devastating bacterium to cause black rot disease in crucifers. To study the genetic diversity and virulence analysis, 24 isolates of Xcc were collected from cole crops including cauliflower, cabbage, broccoli and knol khol from different agro-climatic regions of India ranging from temperate to subtropical climates. For virulence analysis, 24 isolates of Xcc were tested on 27 cultivars of crucifers including seven species of Brassica spp. (B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa), Sinapsis alba, Eruca sativa and Raphanus sativus under field conditions at IARI, New Delhi, during November 2010–March 2011. Maximum disease incidence 85.15% was found in the cultivars of crucifers caused by strains Xcc-C124, Xcc-C6, Xcc-C125, Xcc-C111 and Xcc-C131 after 15 days of inoculation and significantly increased after 30 days. Black rot severity in cultivars of crucifers varied from 0 to 6.9 and 0 to 7.9 out of 9 scale after 15 and 30 days of inoculation, respectively. But, no disease incidence was recorded on all the tested cultivars of B. juncea (Pusa Bold, Varuna, Pusa Mustard-21 and Pusa Vijay) against all the strains of Xcc after 15 days. Genetic diversity of 24 strains of Xcc was studied using REP- and BOX-PCR, indicating the existence of wide range of genetic diversity among the strains. The strains were clustered into two groups at 50% similarity coefficient and designated as Group 1 and Group 2. The majority of the strains (23 strains) were clustered under Group 1 except Xcc-C120, which formed separate group (Group 2). In the present study, genetic diversity and virulence pattern in Indian strains of Xcc were established, which will be helpful in the development of resistant genotypes against this bacterial pathogen.     

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra‐pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc ‐ Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max‐exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT‐PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.     

Genetic Diversity and Population Structure of the Xanthomonas campestris pv. campestris Strains Affecting Cabbages in China Revealed by MLST and Rep-PCR Based Genotyping

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.     

Plant responses underlying nonhost resistance of Citrus limon against Xanthomonas campestris pv. campestris

Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by Xanthomonas citri ssp. citri (X. citri); thus, new sustainable strategies to manage this disease are needed. Although all Citrus spp. are susceptible to this pathogen, they are resistant to other Xanthomonas species, exhibiting non-host resistance (NHR), for example, to the brassica pathogen X. campestris pv. campestris (Xcc) and a gene-for-gene host defence response (HDR) to the canker-causing X. fuscans ssp. aurantifolii (Xfa) strain C. Here, we examine the plant factors associated with the NHR of C. limon to Xcc. We show that Xcc induced asymptomatic type I NHR, allowing the bacterium to survive in a stationary phase in the non-host tissue. In C. limon, this NHR shared some similarities with HDR; both defence responses interfered with biofilm formation, and were associated with callose deposition, induction of the salicylic acid (SA) signalling pathway and the repression of abscisic acid (ABA) signalling. However, greater stomatal closure was seen during NHR than during HDR, together with different patterns of accumulation of reactive oxygen species and phenolic compounds and the expression of secondary metabolites. Overall, these differences, independent of Xcc type III effector proteins, could contribute to the higher protection elicited against canker development. We propose that Xcc may have the potential to steadily activate inducible defence responses. An understanding of these plant responses (and their triggers) may allow the development of a sustained and sustainable resistance to citrus canker.     

Real Time Live Imaging of Phytopathogenic Bacteria Xanthomonas campestris pv. campestris MAFF106712 in ‘Plant Sweet Home’

Xanthomonas is one of the most widespread phytobacteria, causing diseases on a variety of agricultural plants. To develop novel control techniques, knowledge of bacterial behavior inside plant cells is essential. Xanthomonas campestris pv. campestris, a vascular pathogen, is the causal agent of black rot on leaves of Brassicaceae, including Arabidopsis thaliana. Among the X. campestris pv. campestris stocks in the MAFF collection, we selected XccMAFF106712 as a model compatible pathogen for the A. thaliana reference ecotype Columbia (Col-0). Using modified green fluorescent protein (AcGFP) as a reporter, we observed real time XccMAFF106712 colonization in planta with confocal microscopy. AcGFP-expressing bacteria colonized the inside of epidermal cells and the apoplast, as well as the xylem vessels of the vasculature. In the case of the type III mutant, bacteria colonization was never detected in the xylem vessel or apoplast, though they freely enter the xylem vessel through the wound. After 9 days post inoculation with XccMAFF106712, the xylem vessel became filled with bacterial aggregates. This suggests that Xcc colonization can be divided into main four steps, (1) movement in the xylem vessel, (2) movement to the next cell, (3) adhesion to the host plant cells, and (4) formation of bacterial aggregates. The type III mutant abolished at least steps (1) and (2). Better understanding of Xcc colonization is essential for development of novel control techniques for black rot.     

A novel 3-oxoacyl-ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants.     

The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A TOOLBOX FOR SEQUENTIAL PLANT N-GLYCAN PROCESSING*

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N₂M₃FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.     

Regulation of Cell Wall-Bound Invertase in Pepper Leaves by Xanthomonas campestris pv. vesicatoria Type Three Effectors

Xanthomonas campestris pv. vesicatoria (Xcv) possess a type 3 secretion system (T3SS) to deliver effector proteins into its Solanaceous host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that Xcv may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient Xcv strain showed a higher level of cw-Inv mRNA and enzyme activity relative to Xcv wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the pathogenesis-related gene PRQ. These results suggest that Xcv suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify Xcv T3Es that regulate cw-Inv activity, a screen was performed with eighteen Xcv strains, each deficient in an individual T3E. Seven Xcv T3E deletion strains caused a significant change in cw-Inv activity compared to Xcv wild type. Among them, Xcv lacking the xopB gene (Xcv ΔxopB) caused the most prominent increase in cw-Inv activity. Deletion of xopB increased the mRNA abundance of PRQ in Xcv ΔxopB-infected pepper leaves, but not of Pti5 and Acre31, two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited Xcv-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity in planta to suppress sugar-enhanced defense responses during Xcv infection.     

Identification of genes differentially expressed in cauliflower associated with resistance to <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>

Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is one of the most damaging diseases of cauliflower and other crucifers. In order to investigate the molecular resistance mechanisms and to find the genes related to black rot resistance in cauliflower, a suppression subtractive hybridization (SSH) cDNA library was constructed using resistant line C712 and its susceptible near-isogenic line C731 as tester and driver, respectively. A total of 280 clones were obtained from the library by reverse northern blotting. Sequencing analysis and homology searching showed that these clones represent 202 unique sequences. The library included many defense/disease-resistant related genes, such as plant defensin gene PDF1.2, lipid transfer protein, thioredoxin h. Gene expression profiles of 12 genes corresponding to different functional categories were monitored by real-time RT-PCR. The results showed that the expression induction of these genes in the susceptible line C712 in response to Xcc was quicker and more intense, while in C731 the reaction was delayed and limited. Our results imply that these up-regulated genes might be involved in cauliflower responses against Xcc infection. Information obtained from this study could be used to understand the molecular mechanisms of disease response in cauliflower under Xcc stress.