Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc–Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label‐free shotgun 2D‐nanoUPLC/MS^E to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc–susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large‐scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant–pathogen interaction in planta.     

Using antagonistic soil bacteria and their cell‐free filtrates to control the black rot pathogen Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacteria, and it is the causative agent of black rot in crucifers. Recent studies have shown that Bacillus species have strong biological control on Xanthomonas. One of the mechanisms of this control is secondary metabolites production. A collection of 257 bacteria isolated from a suppressive soil was evaluated for in vitro antagonistic activity against X. campestris, and 92 isolates (44.6%) were able to inhibit its growth. Among the 92 isolates evaluated in the double‐layer technique, 51 (55.43%) inhibited Xcc growth on the inhibition tests with cell‐free filtrates (CFF) in liquid medium. Thirteen of these isolates presented 50% or more growth inhibition, and five isolates presented 100% growth inhibition of Xcc. The CFF of the isolate TCDT‐08, which belongs to the Paenibacillus genus, was used for in vivo tests with kale crops. The artificial inoculation of kale with Xcc‐629IBSBF pretreated with CFF from the isolate TCDT‐08 demonstrated that the bacterium loses the ability of colonizing kale and of causing black rot. A Paenibacillus sp. isolate has strong inhibitory activity against X. campestris pv. campestris, and further studies can result in the use of this isolate to protect kale from Xcc infection.     

Xanthomonas campestris sensor kinase HpaS co-opts the orphan response regulator VemR to form a branched two-component system that regulates motility

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.     

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.     

Genetic and Pathogenic Variability of Indian Strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> Causing Black Rot Disease in Crucifers

Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) causing black rot of crucifers is a serious disease in India and causes >50% crop losses in favorable environmental conditions. Pathogenic variability of Xcc, X. oryzae pv. oryzae (Xoo), and X. axonopodis pv. citri (Xac) were tested on 19 cultivars of cruciferae including seven Brassica spp. viz., B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa, and Raphanus sativus for two consecutive years viz., 2007–2008 and 2008–2009 under field conditions at Indian Agricultural Research Institute, New Delhi. Xcc (22 strains) and other species of Xanthomonas (2 strains), they formed three distinct groups of pathogenic variability i.e., Group 1, 2, and 3 under 50% minimum similarity coefficient. All strains of Xcc clustered under Groupl except Xcc-C20. The strains of Xcc further clustered in 6 subgroups viz., A, B, C, D, E, and F based on diseases reaction on host. Genetic variability of 22 strains of Xcc was studied by using Rep-PCR (REP-, BOX- and ERIC-PCR) and 10 strains for hrp (hypersensitive reaction and pathogenecity) gene sequence analysis. Xcc strains comprised in cluster 1, Xac under cluster 2, while Xoo formed separate cluster 3 based on >50% similarity coefficient. Cluster 1 was further divided into 8 subgroups viz., A, B, C, D, E, F, G, and H at 75% similarity coefficient. The hrpF gene sequence analysis also showed distinctness of Xcc strains from other Xanthomonads. In this study, genetic and pathogenic variability in Indian strains of Xcc were established, which will be of immense use 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.     

Karyotype and genomic in situ hybridization pattern in ×Brassicoraphanus, an intergeneric hybrid between Brassica campestris ssp. pekinensis and Raphanus sativus

The karyotype and genomic in situ hybridization (GISH) of an intergeneric hybrid Baemoochae, ×Brassicoraphanus, which originated from hybridization between Chinese cabbage, Brassica campestris (synonym, rapa) ssp. pekinensis, and radish, Raphanus sativus, were analyzed to determine its chromosome complement. In the karyotype analysis, B. campestris was verified to have 2n = 20 chromosomes, including a particular pair of the subtelomeric chromosomes with the nucleolar organizer; R. sativus to have 2n = 18 chromosomes, including a particular pair of the submetacentric chromosomes with the secondary constriction of nucleolar organizer; and ×Brassicoraphanus to have 2n = 38 chromosomes, including both the subtelomeric chromosomes of Brassica and the secondary constriction chromosome pair of Raphanus. These findings indicate that ×Brassicoraphanus is a polyploid between Brassica and Raphanus. In the GISH analysis using chromosomes of B. campestris and R. sativus as the probe and blocking DNA, respectively, only 20 chromosomes of Brassica had hybridization signals. This result reveals that ×Brassicoraphanus is an intergeneric hybrid consisting of the complete genomes of both Brassica and Raphanus. However, the nucleolar organizers of Brassica and Raphanus were not identified because the hybridization signals appeared to be centering mainly around the centromere, becoming weak at the edges.     

Characterization of <Emphasis Type="Italic">Campylobacter</Emphasis> phages including analysis of host range by selected <Emphasis Type="Italic">Campylobacter</Emphasis> Penner serotypes

Background  

The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans.     

Fraction I protein analysis of parasexual hybrid plants Arabidopsis thaliana + Brassica campestris

The polypeptide composition of Fraction I protein (ribulose-1,5-bisphosphate carboxylase) prepared from leaves of two clones of the parasexual hybrid plant Arabidopsis thaliana + Brassica campestris as well as their parents was analyzed by isoelectric focusing. The protein in hybrid plants contained a heterogenous population of small subunits resulting from the expression of both Arbabidopsis and Brassica nuclear genes, whereas the large subunit polypeptides, and hence the functional chloroplast DNA, were from the Brassica parent.     

Inheritance of isozyme and RFLP markers in Brassica campestris and comparison with B. oleracea

Summary Using primarily cDNA restriction fragment length polymorphism markers (RFLPs) previously located to Brassica oleracea (cabbage, 2n=18) chromosomes, we initiated a comparative RFLP map in an F₂ population of B. campestris (turnip x mock pak-choi, 2n=20). As with B. oleracea, the genome of B. campestris showed extensive gene duplication, and the majority of detected duplicated loci were unlinked. Only 6 of the 49 identified loci were represented as a single copy, and 3 of these 6 were clustered on a single linkage group showing a distorted segregation ratio. Comparison with B. Oleracea indicates this synteny is conserved between species. Two other linkage groups also appeared syntenic between B. oleracea and B. campestris. One single copy locus appears to have changed synteny between B. oleracea and B. campestris. These observations suggest that B. oleracea and B. campestris share a common ancestor, but that chromosome repatterning has occurred during or after speciation. Within B. campestris, 5 loci appeared duplicated in one parent or the other, and 2 of these were linked. Differentiation through subspecies-specific duplication or deletion events is suggested as one mechansim for the evolution of numerous morphotypes within each of these species.     

Novel broad-spectrum bacteriophages against Xanthomonas oryzae and their biocontrol potential in rice bacterial diseases

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS)—caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively—are two major bacterial diseases that threaten the safe production of rice, one of the most important food crops. Bacteriophages are considered potential biocontrol agents against rice bacterial pathogens, due to their host specificity and environmental safety. It is common for BLB and BLS to occur together in fields, which highlights the need for broad-spectrum phages capable of infecting both Xoo and Xoc. In this study, two lytic broad-spectrum phages (pXoo2106 and pXoo2107) that can infect various strains of Xoo and Xoc were assessed. Both phages belong to the class Caudoviricetes and one of them to the family Autographiviridae, while the other belongs to an unclassified family. Two phages alone or combined in a phage cocktail could effectively inhibit Xoo and Xoc growth in vitro. In an in vivo biocontrol experiment, the phage cocktail reduced the total CFU and significantly eased the symptoms caused by Xoo or Xoc. Our results suggest that pXoo2106 and pXoo2107 have a broad-spectrum host range targeting different X. oryzae strains, and have strong biocontrol potential in field applications against both BLB and BLS.     

A comparative study of DNA synthesis in cotyledon protoplast cultures of Brassica napus,B. campestris and B. oleracea using an immunocytochemical technique

The present study was designed (1) to observe the characterization of 5-bromo-2′-dexoyuridine (BrdU) incorporation into cultured Brassica cotyledon protoplasts and (2) to investigate the genetic differences in the levels of nuclear DNA synthesis (expressed by the percentage of nuclei labelled with BrdU) in cotyledon protoplast cultures from 12 cultivars of three Brassica species (Brassica napus, B. campestris and B. oleracea) at an early stage using immunocytochemistry. Nuclei labelled with BrdU were different from those showing only staining with 4′-6′-diamidino-2-phenylindole (DAPI) under fluorescence and light microscopy. Two to 5% of nuclei were labelled with BrdU after 1 h of culture, indicating that nuclear DNA synthesis occurred at a very early stage of culture. The percentage of nuclei labelled with BrdU increased with time over the length of the culture period. The mean percentage of nuclei labelled with BrdU in the 12 cultivars was about 25% at 24 h after culture initiation. The curve of the increase in percentage of nuclei labelled with BrdU exhibited an S-shape from 1 to 24 h. However, cultivar differences in percentages of nuclei labelled with BrdU were very significant over the time course of 1-24 h from initial culture, with cultivars Eureka (B. napus), Global (B. napus), Narc 82 (B. napus), Bunyip (B. campestris) and Sugar Loaf (B. oleracea) having a consistently higher percentage of nuclei labelled with BrdU than the other cultivars. Species differences were also significant, with cultivars of B. napus showing much higher percentages than the tested cultivars of B. campestris and B. oleracea. The results indicate that the differences in nuclear DNA synthesis in Brassica cotyledon protoplast cultures were most likely at both intra- and interspecies levels.