Xanthomonas Campestris Pv Research Articles

The Role of RelA and SpoT on ppGpp Production, Stress Response, Growth Regulation, and Pathogenicity in Xanthomonas campestris pv. campestris.

ABSTRACTThe alarmone ppGpp plays an important role in the survival of bacteria by triggering the stringent response when exposed to environmental stress. Although Xanthomonas campestris pv. campestris (Xcc), which causes black rot disease in crucifers, is a representative species of Gram-negative phytopathogenic bacteria, relatively little is known regarding the factors influencing the stringent response in this species. However, previous studies in other Gram-negative bacteria have indicated that RelA and SpoT play a critical role in ppGpp synthesis. The current study found that these proteins also had an important role in Xcc, with a ΔrelAΔspoT double mutant being unable to produce ppGpp, resulting in changes to phenotype including reduced production of exopolysaccharides (EPS), exoenzymes, and biofilm, as well the loss of swarming motility and pathogenicity. The ppGpp-deficient mutant also exhibited greater sensitivity to environment stress, being almost incapable of growth on modified minimal medium (mMM) and having a much greater propensity to enter the viable but nonculturable (VBNC) state in response to oligotrophic conditions (0.85% NaCl). These findings much advance our understanding of the role of ppGpp in the biology of Xcc and could have important implications for more effective management of this important pathogen.IMPORTANCE Xanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc. Most intriguingly, we found that ppGpp levels and downstream ppGpp-dependent phenotypes were mediated predominantly by SpoT, with RelA having only a supplementary role. Taken together, the results of the current study provide new insight into the role of ppGpp in the biology of Xcc, which could also have important implications for the role of ppGpp in the survival and pathogenicity of other pathogenic bacteria.

Alternative methods for angular stain control in mangoes (Mangifera indica L.)

The conventional methods used for angular stain control are generally chemical methods, however the use of these products can cause high environmental impact and damage to consumer health if it is used in large quantities and undiluted and applied correctly. Based on this problem, this work aimed to evaluate in vitro alternative forms of control using Saccharomyces yeast (with probiotic potential), ethanolic extracts of Mauritia flexuosa (Buriti) and Miconia albicans (Cinnamon-old) plants. To evaluate four GRAS substances in angular leaf spot control caused by Xanthomonas campestris pv. Mangifera indica, during the postharvest period in mangoes. In vitro results using antagonist yeasts showed no inhibitory effect against X. campestris. However, the extracts of the plants Miconia albicans and Mauritia flexuosa showed a significant inhibition. Thus, as two GRAS substances, 1%, 1.5% and 3% sodium carbonate and 3% sodium bicarbonate inhibited X. campestris growth 100%. Given the results obtained, the plant extracts and the GRAS substances tested were effective in controlling phytobacteria and proved to be an alternative in controlling angular leaf spot, thus avoiding economic losses during the mango postharvest phase.

Early Defense Mechanisms of Brassica oleracea in Response to Attack by Xanthomonas campestris pv. campestris.

Black rot disease, caused by Xanthomonas campestris pv. campestris (Xcc), results in significant yield losses in Brassica oleracea crops worldwide. To find black rot disease-resistant cabbage lines, we carried out pathogenicity assays using the scissor-clipping method in 94 different B. oleracea lines. By comparing the lesion areas, we selected a relatively resistant line, Black rot Resistance 155 (BR155), and a highly susceptible line, SC31. We compared the two cabbage lines for the Xcc-induced expression pattern of 13 defense-related genes. Among them, the Xcc-induced expression level of PR1 and antioxidant-related genes (SOD, POD, APX, Trx H, and CHI) were more than two times higher in BR155 than SC31. Nitroblue tetrazolium (NBT) and diaminobenzidine tetrahydrochloride (DAB) staining analysis showed that BR155 accumulated less Xcc-induced reactive oxygen species (ROS) than did SC31. In addition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays showed that BR155 had higher antioxidant activity than SC31. This study, focused on the defense responses of cabbage during the early biotrophic stage of infection, indicated that Xcc-induced ROS might play a role in black rot disease development. We suggest that non-enzymatic antioxidants are important, particularly in the early defense mechanisms of cabbage against Xcc.

The RavA/VemR two-component system plays vital regulatory roles in the motility and virulence of Xanthomonas campestris.

Xanthomonas campestris pv. campestris (Xcc) can cause black rot in cruciferous plants worldwide. Two‐component systems (TCSs) are key for bacterial adaptation to various environments, including hosts. VemR is a TCS response regulator and crucial for Xcc motility and virulence. Here, we report that RavA is the cognate histidine kinase (HK) of VemR and elucidate the signalling pathway by which VemR regulates Xcc motility and virulence. Genetic analysis showed that VemR is epistatic to RavA. Using bacterial two‐hybrid experiments and pull‐down and phosphorylation assays, we found that RavA can interact with and phosphorylate VemR, suggesting that RavA is the cognate HK of VemR. In addition, we found that RpoN2 and FleQ are epistatic to VemR in regulating bacterial motility and virulence. In vivo and in vitro experiments demonstrated that VemR interacts with FleQ but not with RpoN2. RavA/VemR regulates the expression of the flagellin‐encoding gene fliC by activating the transcription of the rpoN2‐vemR‐fleQ and flhF‐fleN‐fliA operons. In summary, our data show that the RavA/VemR TCS regulates FleQ activity and thus influences the expression of motility‐related genes, thereby affecting Xcc motility and virulence. The identification of this novel signalling pathway will deepen our understanding of Xcc–plant interactions.

Xanthomonas transcriptome inside cauliflower hydathodes reveals bacterial virulence strategies and physiological adaptations at early infection stages.

Xanthomonas campestris pv. campestris (Xcc) is a seed‐transmitted vascular pathogen causing black rot disease on cultivated and wild Brassicaceae. Xcc enters the plant tissues preferentially via hydathodes, which are organs localized at leaf margins. To decipher both physiological and virulence strategies deployed by Xcc during early stages of infection, the transcriptomic profile of Xcc was analysed 3 days after entry into cauliflower hydathodes. Despite the absence of visible plant tissue alterations and despite a biotrophic lifestyle, 18% of Xcc genes were differentially expressed, including a striking repression of chemotaxis and motility functions. The Xcc full repertoire of virulence factors had not yet been activated but the expression of the HrpG regulon composed of 95 genes, including genes coding for the type III secretion machinery important for suppression of plant immunity, was induced. The expression of genes involved in metabolic adaptations such as catabolism of plant compounds, transport functions, sulphur and phosphate metabolism was upregulated while limited stress responses were observed 3 days postinfection. We confirmed experimentally that high‐affinity phosphate transport is needed for bacterial fitness inside hydathodes. This analysis provides information about the nutritional and stress status of bacteria during the early biotrophic infection stages and helps to decipher the adaptive strategy of Xcc to the hydathode environment.

First report of Xanthomonas campestris pv. campestris as the causal agent of necrotic leaf spot in Phaseolus vulgaris at Puebla, Mexico.

Beans are the most cultivated legume in the world. In Mexico, it is the second most important crop after corn (FAO 2020; SIAP 2020). Bean plants "Flor de Mayo M38" variety were affected by a foliar disease during the agricultural cycle 2019 in Puebla-Mexico (19°02'46.6" LN and 98°05'15.6" LO). Necrotic V- shaped lesions were observed on the margins of the leaves surrounded by yellow halos followed by foliar necrosis, affecting 40% of the crop. In Mexico this variety of cultivars is in great demand for local consumption and generates income in foreign currency (Castellanos et al. 1997). Sampling was carried out on 50 plants "Flor de Mayo M38" variety, with necrotic leaf symptoms from ten plots of one hectare. Samples were cut into pieces (5 mm), disinfested with 1% hypochlorite 3 min, and washed with sterile distilled water. Subsequently, samples were dried on sterile paper and placed on Petri plates containing yeast extract calcium carbonate dextrose agar (YDC) medium and kept at 36°C for 3 days. Colonies of ten typical bacteria isolated from all symptomatic plants were Gram (-), small and uniform in size with rounded edges, yellow, convex with entire borders and mucoid appearance on YDC. Bacteria did not grow on 0.1% triphenyl tetrazolium chloride amended casamino acid, peptone, and glucose medium (CPG). Biochemical tests showed that isolates did not reduce nitrate to nitrites, had positive catalase and starch hydrolysis, while the Kovac oxidase test was negative (Schaad and White 1974). Genus identity of the representative isolate Xcf1-APJR, was confirmed by 16S rRNA encoding gene partial sequencing, using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5'-TACCAGGGTATCTAATCC-3') (Halim et al. 2020). BLASTn alignments against the nucleotide collection were 100% identical to Xanthomonas sequences including Xanthomonas campestris pv. campestris strains NZ_AP019684.1, CP025750.1, and MN108237.1. The 1,418 bp sequence was deposited in the GenBank database under accession number MT645246. The identification of species/pathovar was accomplished by serological methods using a polyclonal antiserum specific for X. campestris pv. campestris (Popovic ́ et al. 2013) with the DAS-ELISA commercial kit (catalog number 07122C/096, LOEWE Biochemica GmbH, Germany). The pathogenicity test was carried out on 50 healthy bean plants from the "Flor de Mayo M38" variety. Bacterial culture incubated at 28°C for 48 h in YDC medium was used to prepare the bacterial suspension (108 CFU mL-1). The first two lower leaves of 30-day-old plants were inoculated by sprinkling. Ten plants sprayed with sterile distilled water were used as negative control. All plants were kept for 20 days in greenhouse at 18-26°C and relative humidity of 60%. After seven days, chlorotic lesions developed on all inoculated plants that became necrotic from 14 days after inoculation (dai). Necrotic leaf spots merged at 14 dai to form necrotic areas of more than 20 mm in diameter, reaching total necrosis of the leaf tissue at 20 dai and were similar to the symptoms observed in the field. Koch's postulates were confirmed by the reisolation of Xcf1-APJR strain, which presented the same colony morphology, partial sequence, and polyclonal specific detection. This is the first report of this pathogen causing necrotic leaf spot in beans from the "Flor de Mayo M38" variety in Puebla-Mexico. The author(s) declare no conflict of interest.

Potentiation of plant defense by bacterial outer membrane vesicles is mediated by membrane nanodomains.

Outer membrane vesicles (OMVs) are released from the outer membranes of Gram-negative bacteria during infection and modulate host immunity during host-pathogen interactions. The mechanisms by which OMVs are perceived by plants and affect host immunity are unclear. Here, we used the pathogen Xanthomonas campestris pv. campestris to demonstrate that OMV-plant interactions at the Arabidopsis thaliana plasma membrane (PM) modulate various host processes, including endocytosis, innate immune responses, and suppression of pathogenesis by phytobacteria. The lipid phase of OMVs is highly ordered and OMVs directly insert into the Arabidopsis PM, thereby enhancing the plant PM's lipid order; this also resulted in strengthened plant defenses. Strikingly, the integration of OMVs into the plant PM is host nanodomain- and remorin-dependent. Using coarse-grained simulations of molecular dynamics, we demonstrated that OMV integration into the plant PM depends on the membrane lipid order. Our computational simulations further showed that the saturation level of the OMV lipids could fine-tune the enhancement of host lipid order. Our work unraveled the mechanisms underlying the ability of OMVs produced by a plant pathogen to insert into the host PM, alter host membrane properties, and modulate plant immune responses.

ITRAQ proteomics reveals the regulatory response to Xanthomonas campestris pv. vesicatoria in resistant vs. susceptible pepper genotypes

Bacterial spot (BS) is a severe bacterial disease induced by Xanthomonas campestris pv. vesicatoria (Xcv), a pathogen that causes serious damage to pepper growth and yield. It is therefore important to study the mechanisms of pepper resistance to Xcv and to breed and promote Xcv-resistant pepper varieties. However, studies of the responses to Xcv infection in peppers at the protein level are limited. Here, we examined Xcv-induced proteomic changes in leaves of the BS susceptible bell pepper ECW and the resistant bell pepper VI037601 using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. A total of 6,120 distinct proteins were identified, and there were 1,289 significantly differentially accumulated proteins (DAPs) in ECW and VI037601 leaves after Xcv inoculation. Among these, 339 (250 up- and 89 down-regulated) and 479 (300 up- and 179 down-regulated) DAPs were specifically identified in ECW and VI037601, respectively, with 459 (364 up- and 95 down-regulated) similarly expressed DAPs being shared by ECW and VI037601. Based on bioinformatics analysis, many defense-associated proteins were identified as up-regulated in ECW and VI037601, especially the proteins involved in plant-pathogen interaction, phenylpropanoid biosynthesis, protein processing in the endoplasmic reticulum, and MAPK signaling pathway - plant. Moreover, we evaluated transcript levels of six differentially expressed genes from the iTRAQ results by qRT-PCR. The analysis revealed transcriptional changes that were consistent with the changes at the protein level. This study will provide a valuable resource for understanding the molecular basis of pepper resistance to Xcv infection and for improving the disease resistance of pepper cultivars.

Current Situation of Mango Bacterial Black Spot Caused by <i>Xanthomonas campestris</i> pv <i>mangiferaeindicae</i> in the Poro and Tchologo Regions in Côte d'Ivoire

Mango bacterial black spot is an emerging disease in Cote d'Ivoire and the damage caused is becoming more and more significant. This study aims at diagnosing bacterial black spot incidence and severity level in regions of intensive mango production. Surveys were carried out during the vegetative stage of mango trees from July to August 2019 and the fruiting stage from March to April 2020 in 50 orchards in the Poro and Tchologo regions. In each orchard, 5 mango trees regularly dispersed over an area of 0.5 ha were assessed. On each tree, three (3) twigs per cardinal point were selected then the leaves counted and the leaf area covered by bacterial black spot symptoms determined. At fruiting stage, bacterial black spot severity on 40 fruits per selected tree, at the rate of 10 fruits per cardinal point was assessed. Bacterial black spot symptoms were observed in all orchards and the bacterium, Xanthomonas campestris pv. Mangiferaeindicae, was systematically isolated basing on symptoms taken from the leaves and fruits collected. Statistical analysis of the mean incidence and severity per town revealed a variability of 83.2% up to 90% on the leaves and 41.42% up to 55% on the fruits regarding incidence. As for severity, the highest values, 10.58% and 5.23% on leaves and fruits respectively were recorded in the department of Tafiere. Mango bacterial black spot is found in orchards located in the Poro and Tchologo regions. Incidence and severity are strongly related to the humidity conditions of the production area.

Molecular and phenotypic characterization of Xanthomonas campestris pv. campestris causing black rot in Brassica crops in Brazil

Molecular and phenotypic characterization of Xanthomonas campestris pv. campestris causing black rot in Brassica crops in Brazil

Efficacy of Fungicides against Soil Borne and Grapevine Pathogens under In vitro Conditions

In vitro bio-efficacy of the novel copper (NC101 and NCP102) and phosphonate (PN103 and PMN104) based fungicides against various soil borne and grape vine pathogens was evaluated at ICAR-National Research Centre for Grapes, Pune, Maharashtra, India during February - April 2021. The fungicides were screened against five bacterial pathogens viz. Xanthomonas campestris pv. citri, X. campestris pv. campestris, X. campestris pv. punicae, X. campestris pv. viticola and X. oryzae pv. oryzae and 10 fungal pathogens viz. Rhizoctonia bataticola, Sclerotium rolfsii, Cladosporium sp, Alternaria alternata, Colletotrichum gloeosporioides, Penicillium notatum, Magnaporthe oryzae, Fusarium oxysporium, Macrophomina phaseolina (Soyabean isolate) and Macrophomina phaseolina (Jute isolate) at different concentrations. Results indicated that among all the tested fungicides viz. NC101, NCP102, PN103 and PMN104, phosphonate based fungicides (PN103 and PMN104) were highly effective against bacterial isolates with zone of inhibition ranging between 8.75 - 31.12 mm in which X. campestris pv. viticola was found to express least inhibition zone. In case of pathogenic fungal isolates, similar trend was observed, most of the isolates showed cent percent inhibition at higher concentration of PN103 and PMN104. However Sclerotium rolfsii showed least or no inhibition when tested at different concentrations of fungicides. The chemicals exhibited wide range of inhibition and it was found to increase steadily with increase in concentrations of the test fungicides.

Anti-bacterial Effect of Aqueous and Ethanolic Extracts of Onion, Garlic and Cinnamon on Xanthomonas Species

Onion (Allium cepa), garlic (Allium sativum) and cinnamon (Cinnamomum zeylanicum) are edible herbs but they contain many chemicals which can be used as medicine. These herbs also contain many antibiotic molecules that are capable to inhibit or reduce growth of many types of microbes including bacteria. Due to the development of resistance of bacteria against synthetic antibiotic now there is a need to have natural antibiotic which must be harmless, cheap and easily available. To know the effectiveness of garlic, onion, and cinnamon on disease causing bacteria such as Xanthomonas campestris pv. malvacearum, Xanthomonas axonopodis pv. citri, Xanthomonas oryzae pv. oryzae, extracts of each sample was made in water and ethanol. Antibacterial potential of plant extracts was observed at two different stages: at the time of inoculation and after growth of pathogen on media. Results showed extracts applied on all the bacteria before growth inhibited maximum bacterial growth as compared to applied after growth. Moreover, cinnamon extract in ethanol effectively control bacterial growth than onion and garlic extracts. Maximum inhibition zone was shown by ethanolic extract of cinnamon before growth of Xanthomonas oryzae pv. oryzae (7.333 mm), Xanthomonas campestris pv. malvacearum (7.83 mm) and Xanthomonas axonopodis pv. citri (5.1 mm). After 24 hours, maximum growth of Xanthomonas oryzae and Xanthomonas axonopodis was inhibited by ethanolic extracts of cinnamon (2.82 and 3.35 mm, respectively) while Xanthomonas campestris was inhibited by ethanolic extract of onion (6.55 mm). Extract diluted from 66mg/ml to 0.01mg/ml showed different minimum inhibitory concentration against pathogens. As concentration decreases inhibition of bacteria also decreases. Ethanolic extracts of cinnamon showed maximum MIC against Xanthomonas campestris pv. malvacearum (3.5 mm to 1.5 mm) and Xanthomonas oryzae pv. oryzae (5 mm to 2 mm) while for Xanthomonas axonopodis pv. citri ethanolic extract of garlic (3 mm to 1 mm) was effective ranging between 66mg/ml to 33 mg/ml. This study highlighted that natural products possess ability to inhibit pathogenic bacterial growth and would also be helpful in medicinal field for further study.

Effects of Moringa oleifera Leaf Extracts on Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium belonging to the Xanthomonodaceae family, causing black rot in crucifers. To control this pathogen, the study investigated the effect of different leaves extracts of Moringa oleifera Lam., a tropical plant, well known for its food properties and with countless applications in many different fields, from nutraceutical (hypoglycemic) to the cosmetic (sunscreen) properties. Nevertheless, several studies pointed to its antibacterial action against both Gram-negative and Gram-positive bacteria. Many bioactive compounds, including flavonoids, phenolic acids, alkaloids, isothiocyanates, tannins and saponins, contained in these extracts, are responsible for its countless activities. The analyses carried out in this study show that the methanolic, hydroalcoholic and hydroalcoholic maltodextrin extracts have both bacteriostatic and bactericidal effects at concentrations of 0.5, 0.5 and 0.1 mg/mL respectively. In particular, the study shows how all extracts can alter membrane permeability, to adversely affect swarming motility, and to alter biofilm formation in Xcc. The in planta experiments showed a reduction of the necrosis area in the infected radishes, although the ability of the extracts to be absorbed by root systems is yet to be understood, in order to reach the target point.

Single Stem Rouging of Banana Xanthomonas Wilt Infected Plants for Orchard Rehabilitation

Banana Xanthomonas Wilt (BXW), caused by Xanthomonas campestris pv. musacearum (Xcm), a devastating disease, causes up to 100% loss and affects all banana varieties. The disease is widespread in East and Central Africa region. Study objective was to evaluate the performance of single stem rouging options for rehabilitation of severely affected banana orchard. Five single stem rouging options were implemented in banana orchard with 80% BXW severity in Western Kenya. The options included rouging of infected banana plants; uprooting, cutting at the base, injecting 20 ml of glyphosate herbicide at the soil base of the pseudostem, uprooting the whole stool and control leaving the infected stools. Initial visual assesment before implementation of the orchard was over 80% infection. Later samples were collected for diagnosis of ten plants per treatment from different plant parts and were subjected to ELISA and PCR procedures for confirmion of the presence of Xcm. Rouging options was implemented for one year. During and after experimentation periodic visual assesment of BXW incidence and severity reduced. Results of ELISA assays and PCR results indicated Xanthomonas was present on banana plants. Rouging the infected banana stems by cutting at the base, uprooting and injection of herbicide reduced BXW incidence within six months. Xanthomonas, an inhibiting bacteria cannot surve without host and by killing the banana plant the Xanthomonas dies. Yields were recovered from the rehabilitated orchard within one year. Banana orchard highly infected with BXW was effectively rehabilitated.

XANTHOMONAS CAMPESTRIS: PRINCIPAL RESULTS AND PROBLEMS IN STUDY OF BRASSICAS BLACK ROT CAUSING AGENT

Black rot (caus. agent Xanthomonas campestris pv. campestris (Dowson) Dye, et al. (1980) is the most harmful disease of brassicas, widespread across the World. Plant breeding for resistance to Xcc received a new push after the discovery a racespecific reaction in several plant species of the Brassicaceae family in 1992 and, accordingly, the racial structure of the pathogen population. The number of pathogen races (11 at the moment) and putative resistance genes is constantly increasing,
 which makes it difficult to breed plants following a complex model describing the gene-to-gene relationship between the pathogen and plants. The developed molecular markers of resistance genes and pathogen races do not yet give a reliable result due to the high genetic diversity of populations. This review identifies the main tasks and suggests possible ways to solve them.

A Bifunctional Synthetic Peptide With Antimicrobial and Plant Elicitation Properties That Protect Tomato Plants From Bacterial and Fungal Infections.

The hybrid peptide BP178 (KKLFKKILKYLAGPAGIGKFLHSAKKDEL-OH), derived from BP100 (KKLFKKILKYL) and magainin (1–10), and engineered for plant expression, had a strong bactericidal activity but not fungicidal. Moreover, the preventive spray of tomato plants with BP178 controlled infections by the plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria, as well as the fungus Botrytis cinerea. The treatment of tomato plants with BP178 induced the expression of several genes according to microarray and RT-qPCR analysis. Upregulated genes coded for several pathogenesis-related proteins, including PR1, PR2, PR3, PR4, PR5, PR6, PR7, PR9, PR10, and PR14, as well as transcription factors like ethylene transcription factors, WRKY, NAC and MYB, involved in the salicylic acid, jasmonic acid, and ethylene-signaling pathways. BP178 induced a similar gene expression pattern to flg15 according to RT-qPCR analysis, whereas the parent peptide BP100 did not trigger such as a strong plant defense response. It was concluded that BP178 was a bifunctional peptide protecting the plant against pathogen infection through a dual mechanism of action consisting of antimicrobial activity against bacterial pathogens and plant defense elicitation on plant host.

The Contribution of the Predicted Sorting Platform Component HrcQ to Type III Secretion in Xanthomonas campestris pv. vesicatoria Depends on an Internal Translation Start Site.

Pathogenicity of the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of at least nine structural core components, which are designated Sct (secretion and cellular translocation) in animal-pathogenic bacteria. Sct proteins are involved in the assembly of the membrane-spanning secretion apparatus which is associated with an extracellular needle structure and a cytoplasmic sorting platform. Components of the sorting platform include the ATPase SctN, its regulator SctL, and pod-like structures at the periphery of the sorting platform consisting of SctQ proteins. Members of the SctQ family form a complex with the C-terminal protein domain, SctQC, which is translated as separate protein and likely acts either as a structural component of the sorting platform or as a chaperone for SctQ. The sorting platform has been intensively studied in animal-pathogenic bacteria but has not yet been visualized in plant pathogens. We previously showed that the SctQ homolog HrcQ from X. campestris pv. vesicatoria assembles into complexes which associate with the T3S system and interact with components of the ATPase complex. Here, we report the presence of an internal alternative translation start site in hrcQ leading to the separate synthesis of the C-terminal protein region (HrcQC). The analysis of genomic hrcQ mutants showed that HrcQC is essential for pathogenicity and T3S. Increased expression levels of hrcQ or the T3S genes, however, compensated the lack of HrcQC. Interaction studies and protein analyses suggest that HrcQC forms a complex with HrcQ and promotes HrcQ stability. Furthermore, HrcQC colocalizes with HrcQ as was shown by fluorescence microscopy, suggesting that it is part of the predicted cytoplasmic sorting platform. In agreement with this finding, HrcQC interacts with the inner membrane ring protein HrcD and the SctK-like linker protein HrpB4 which contributes to the docking of the HrcQ complex to the membrane-spanning T3S apparatus. Taken together, our data suggest that HrcQC acts as a chaperone for HrcQ and as a structural component of the predicted sorting platform.

Gene-Based Markers in Marker-Assisted Selection to Screen Tomato Genotypes Resistant to Fusarium Wilt, Late Blight, Verticillium Wilt, Leaf Mold, Bacterial Spot and Bacterial Speck

The tomato crop is exposed to serious losses due to infection with several diseases and pests, which threaten tomato production in Egypt and worldwide. Therefore, selecting the tomato germplasm resistant to a specific pathogen by molecular markers closely linked to resistance loci is a desirable goal of this study. In this work, seven co-dominant markers targeting six resistance genes (I-1, Ve, Ph3, Cf-9/Cf-4, Rx4, and Pto) for six main diseases [ fusarium wilt (Fusarium oxysporum f. sp. lycopersici), verticillium wilt (Verticillium dahliae and V. alboatrum), late blight (Phytophthora infestans), leaf mold (Cladosporium fulvum), bacterial spot (Xanthomonas campestris pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato)], respectively were determined. Theses molecular markers differentiated among 19 tomato genotypes resistant (homozygote/heterozygote) and susceptible (homozygote) to the pathogens. Therefore, this study supplied us with novel tomato lines with resistance to multiple diseases, and their pyramiding inside domesticated tomato cultivars are suggested to apply in the tomato breeding programs of resistance against fungal and bacterial diseases.

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.

The Same against Many: AtCML8, a Ca2+ Sensor Acting as a Positive Regulator of Defense Responses against Several Plant Pathogens.

Calcium signals are crucial for the activation and coordination of signaling cascades leading to the establishment of plant defense mechanisms. Here, we studied the contribution of CML8, an Arabidopsis calmodulin-like protein in response to Ralstonia solanacearum and to pathogens with different lifestyles, such as Xanthomonas campestris pv. campestris and Phytophtora capsici. We used pathogenic infection assays, gene expression, RNA-seq approaches, and comparative analysis of public data on CML8 knockdown and overexpressing Arabidopsis lines to demonstrate that CML8 contributes to defense mechanisms against pathogenic bacteria and oomycetes. CML8 gene expression is finely regulated at the root level and manipulated during infection with Ralstonia, and CML8 overexpression confers better plant tolerance. To understand the processes controlled by CML8, genes differentially expressed at the root level in the first hours of infection have been identified. Overexpression of CML8 also confers better tolerance against Xanthomonas and Phytophtora, and most of the genes differentially expressed in response to Ralstonia are differentially expressed in these different pathosystems. Collectively, CML8 acts as a positive regulator against Ralstonia solanaceraum and against other vascular or root pathogens, suggesting that CML8 is a multifunctional protein that regulates common downstream processes involved in the defense response of plants to several pathogens.