Search

Goss's bacterial wilt and leaf blight of maize (Zea mays) caused by the gram‐positive coryneform bacterium Clavibacter nebraskensis is an economically important disease in North America. C. nebraskensis is included within the high‐risk list of quarantine pathogens by several plant protection organizations (EPPO code: CORBMI), hence it is under strict quarantine control around the world. The causal agent was reported for the first time on maize in Nebraska (USA) in 1969. After an outbreak during the 1970s, prevalence of the disease decreased in the 1980s to the early 2000s, before the disease resurged causing a serious threat to maize production in North America. The re‐emergence of Goss's wilt in the corn belt of the United States led to several novel achievements in understanding the pathogen biology and disease control. In this review, we provide an updated overview of the pathogen taxonomy, biology, and epidemiology as well as management strategies of Goss's wilt disease. First, a taxonomic history of the pathogen is provided followed by symptomology and host range, genetic diversity, and pathogenicity mechanisms of the bacterium. Then, utility of high‐throughput molecular approaches in the precise detection and identification of the pathogen and the management strategies of the disease are explained. Finally, we highlight the role of integrated pest management strategies to combat the risk of Goss's wilt in the 21st century maize industry. Disease symptoms: Large (2–15 cm) tan to grey elongated oval lesions with wavy, irregular water‐soaked margins on the leaves. The lesions often start at the leaf tip or are associated with wounding caused by hail or wind damage. Small (1 mm in diameter), dark, discontinuous water‐soaked spots, known as "freckles", can be observed in the periphery of lesions. When backlit, the freckles appear translucent. Early infection (prior to growth stage V6) may become systemic and cause seedlings to wilt, wither, and die. Coalescence of lesions results in leaf blighting. Host range: Maize (Zea mays) is the only economic host of the pathogen. A number of Poaceae species are reported to act as secondary hosts for C. nebraskensis. Taxonomic status of the pathogen: Class: Actinobacteria; Order: Micrococcales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter nebraskensis. Synonyms Corynebacterium nebraskense (Schuster, 1970) Vidaver & Mandel 1974; Corynebacterium michiganense pv. nebraskense (Vidaver & Mandel 1974) Dye & Kemp 1977; Corynebacterium michiganense subsp. nebraskense (Vidaver & Mandel 1974) Carlson & Vidaver 1982; Clavibacter michiganense subsp. nebraskense (Vidaver & Mandel 1974) Davis et al. 1984; Clavibacter michiganensis subsp. nebraskensis (Vidaver & Mandel 1974) Davis et al. 1984. Type materials: ATCC 27794T; CFBP 2405T; ICMP 3298T; LMG 3700T; NCPPB 2581T. Microbiological properties: Cells are gram‐positive, orange‐pigmented, pleomorphic club‐ or rod‐shaped, nonspore‐forming, nonmotile, and without flagella, approximately 0.5 × 1–2.0 μm. Distribution: The pathogen is restricted to Canada and the United States. Phytosanitary categorization: EPPO code CORBNE. [ABSTRACT FROM AUTHOR]

The plant pathogen Agrobacterium tumefaciens causes crown gall disease and is a widely used tool for generating transgenic plants owing to its virulence. The pathogenic process involves a shift from an independent to a living form within a host plant. However, comprehensive analyses of metabolites, genes, and reactions contributing to this complex process are lacking. To gain new insights about the pathogenicity from the viewpoints of physiology and cellular metabolism, a genome‐scale metabolic model (GSMM) was reconstructed for A. tumefaciens. The model, referred to as iNX1344, contained 1,344 genes, 1,441 reactions, and 1,106 metabolites. It was validated by analyses of in silico cell growth on 39 unique carbon or nitrogen sources and the flux distribution of carbon metabolism. A. tumefaciens metabolic characteristics under three ecological niches were modelled. A high capacity to access and metabolize nutrients is more important for rhizosphere colonization than in the soil, and substantial metabolic changes were detected during the shift from the rhizosphere to tumour environments. Furthermore, by integrating transcriptome data for tumour conditions, significant alterations in central metabolic pathways and secondary metabolite metabolism were identified. Overall, the GSMM and constraint‐based analysis could decode the physiological and metabolic features of A. tumefaciens as well as interspecific interactions with hosts, thereby improving our understanding of host adaptation and infection mechanisms. [ABSTRACT FROM AUTHOR]

Summary: The bacterial stringent response is a response to nutrition deprivation and other stress conditions. In Gram‐negative bacteria, this process is mediated by the small signal molecules guanosine pentaphosphate pppGpp and guanosine tetraphosphate ppGpp (collectively referred to as (p)ppGpp), and the RNA polymerase‐binding transcription factor DksA. The (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT are responsible for cellular (p)ppGpp levels. Here, we investigated the roles of DksA and (p)ppGpp in the virulence traits of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker. ΔdksA and (p)ppGpp‐deficient ΔspoTΔrelA strains caused reduced virulence and compromised growth in host plants, indicating that DksA and (p)ppGpp are required for full virulence of Xcc. To characterize the effect of stringent response regulators on gene expression, RNA‐seq was conducted using ΔdksA and ΔspoTΔrelA mutant strains grown in hrp‐inducing XVM2 medium. Transcriptome analyses showed that DksA and (p)ppGpp repressed the expression of genes encoding tRNAs, ribosome proteins, iron acquisition and flagellum assembly, and enhanced the expression of genes for histidine metabolism, type 3 secretion system (T3SS), type 2 secretion system (T2SS) and TonB‐dependent transporters. Phenotypically, the ΔdksA and ΔspoTΔrelA strains displayed altered motility, enhanced siderophore production and were unable to cause the hypersensitive response on non‐host plants. In conclusion, stringent response regulators DksA and (p)ppGpp play an important role in virulence, nutrition uptake and host adaptation of Xcc. [ABSTRACT FROM AUTHOR]

Summary: Citrus sudden death‐associated virus (CSDaV) is a member of the genus Marafivirus in the family Tymoviridae, and has been associated with citrus sudden death (CSD) disease in Brazil. Difficulties in the purification of CSDaV from infected citrus plants have prevented progress in the investigation of the role of this virus in CSD and an understanding of its molecular biology. In this work, we have constructed a full‐length cDNA clone of CSDaV driven by the 35S promoter (35SRbz‐CSDaV). Agrobacterium tumefaciens‐mediated inoculation of 35SRbz‐CSDaV in Nicotiana benthamiana plants enabled a fast recovery of large amounts of virions from the agroinfiltrated leaves, which allowed a better molecular characterization of CSDaV. In vivo analyses of mutant versions of 35SRbz‐CSDaV revealed the expression strategies used by CSDaV for production of the capsid proteins (CPs). We showed that CSDaV virions contain three forms of CP, each of which is generated from the same coding sequence, but by different mechanisms. The major CPp21 is a product of direct translation by leaky scanning from the second start codon in the subgenomic RNA (sgRNA), whereas the minor CPs, p25 and p23, are produced by direct translation from the first start codon in the sgRNA and by trans‐proteolytic cleavage processing derived from the p25 precursor, respectively. Together, these findings contribute to advance our understanding of CSDaV genome expression strategies. In addition, the construction and characterization of the CSDaV infectious clone represent important steps towards the investigation of the role of this virus in CSD and of its use as a tool for citrus biotechnology. [ABSTRACT FROM AUTHOR]

Summary: The rice XA21‐mediated immune response is activated on recognition of the RaxX peptide produced by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The 60‐residue RaxX precursor is post‐translationally modified to form a sulfated tyrosine peptide that shares sequence and functional similarity with the plant sulfated tyrosine (PSY) peptide hormones. The 5‐kb raxX‐raxSTAB gene cluster of Xoo encodes RaxX, the RaxST tyrosylprotein sulfotransferase, and the RaxA and RaxB components of a predicted type I secretion system. To assess raxX‐raxSTAB gene cluster evolution and to determine its phylogenetic distribution, we first identified rax gene homologues in other genomes. We detected the complete raxX‐raxSTAB gene cluster only in Xanthomonas spp., in five distinct lineages in addition to X. oryzae. The phylogenetic distribution of the raxX‐raxSTAB gene cluster is consistent with the occurrence of multiple lateral (horizontal) gene transfer events during Xanthomonas speciation. RaxX natural variants contain a restricted set of missense substitutions, as expected if selection acts to maintain peptide hormone‐like function. Indeed, eight RaxX variants tested all failed to activate the XA21‐mediated immune response, yet retained peptide hormone activity. Together, these observations support the hypothesis that the XA21 receptor evolved specifically to recognize Xoo RaxX. [ABSTRACT FROM AUTHOR]

Summary: Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone‐like protein, homologue to the β‐subunit of the Heat‐Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild‐type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild‐type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone‐like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum. [ABSTRACT FROM AUTHOR]

Summary : Nicotiana benthamiana is an important model plant for plant–microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG‐tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA‐ or Agrobacterium‐infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r‐proteins). The N. benthamiana riboproteome revealed approximately 6600 r‐protein hits representing 424 distinct r‐proteins that were members of 71 of the expected 81 r‐protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r‐protein composition. In PVA‐infected plants, the number of identified r‐protein paralogues was lower than in Agrobacterium‐infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA‐infected plants co‐purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease‐VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen‐infected N. benthamiana will be useful for studies on the specific use of r‐protein paralogues to control translation in infected plants. [ABSTRACT FROM AUTHOR]

Summary: Xanthomonas citri ssp. citri, a polar flagellated bacterium, causes citrus canker disease worldwide. In this study, we found that the X. citri ssp. citri response regulator VemR plays a regulatory role in flagellum‐derived cell motility. Deletion of the vemR gene resulted in a reduction in cell motility, as well as reductions in virulence and exopolysaccharide production. Reverse transcription‐polymerase chain reaction (RT‐PCR) demonstrated that vemR is transcribed in an operon together with rpoN2 and fleQ. In the vemR mutant, the flagellar distal rod gene flgG was significantly down‐regulated. Because flgG is also rpoN2 dependent, we speculated that VemR and RpoN2 physically interact, which was confirmed by yeast two‐hybrid and maltose‐binding protein (MBP) pull‐down assays. This suggested that the transcription of flgG is synergistically controlled by VemR and RpoN2. To confirm this, we constructed a vemR and rpoN2 double mutant. In this mutant, the reductions in cell motility and flgG transcription were unable to be restored by the expression of either vemR or rpoN2 alone. In contrast, the expression of both vemR and rpoN2 together in the double mutant restored the wild‐type phenotype. Together, our data demonstrate that the response regulator VemR functions as an RpoN2 cognate activator to positively regulate the transcription of the rod gene flgG in X. citri ssp. citri. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Abstract: The biotrophic pathogen Ustilago maydis, the causative agent of corn smut disease, infects one of the most important crops worldwide – Zea mays. To successfully colonize its host, U. maydis secretes proteins, known as effectors, that suppress plant defense responses and facilitate the establishment of biotrophy. In this work, we describe the U. maydis effector protein Cce1. Cce1 is essential for virulence and is upregulated during infection. Through microscopic analysis and in vitro assays, we show that Cce1 is secreted from hyphae during filamentous growth of the fungus. Strikingly, Δcce1 mutants are blocked at early stages of infection and induce callose deposition as a plant defense response. Cce1 is highly conserved among smut fungi and the Ustilago bromivora ortholog complemented the virulence defect of the SG200Δcce1 deletion strain. These data indicate that Cce1 is a core effector with apoplastic localization that is essential for U. maydis to infect its host. [ABSTRACT FROM AUTHOR]

Summary: Taxonomic status: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range: Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties: Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution: Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe. [ABSTRACT FROM AUTHOR]

Summary: Xylella fastidiosa is a Gram‐negative bacterial plant pathogen with an extremely wide host range. This species has recently been resolved into subspecies that correlate with host specificity. This review focuses on the status of X. fastidiosa pathogenic associations in plant hosts in which the bacterium is either endemic or has been recently introduced. Plant diseases associated with X. fastidiosa have been documented for over a century, and much about what is known in the context of host–pathogen interactions is based on these hosts, such as grape and citrus, in which this pathogen has been well described. Recent attention has focused on newly emerging X. fastidiosa diseases, such as in olives. Taxonomy: Bacteria; Gammaproteobacteria; family Xanthomonadaceae; genus Xylella; species fastidiosa. Microbiological properties: Gram‐negative rod (0.25–0.35 × 0.9–3.5 μm), non‐flagellate, motile via Type IV pili‐mediated twitching, fastidious. Host range: Xylella fastidiosa has a broad host range that includes ornamental, ecological and agricultural plants belonging to over 300 different species in 63 different families. To date, X. fastidiosa has been found to be pathogenic in over 100 plant species. In addition, it can establish non‐symptomatic associations with many plants as a commensal endophyte. Here, we list the four distinct subspecies of X. fastidiosa and some of the agriculturally relevant diseases caused by them: X. fastidiosa ssp. fastidiosa causes Pierce's disease (PD) of grapevine (Vitis vinifera); X. fastidiosa ssp. multiplex causes almond leaf scorch (ALS) and diseases on other nut and shade tree crops; X. fastidiosa ssp. pauca causes citrus variegated chlorosis (CVC) (Citrus spp.), coffee leaf scorch and olive quick decline syndrome (OQDS) (Olea europaea); X. fastidiosa ssp. sandyi causes oleander leaf scorch (OLS) (Nerium oleander). Significant host specificity seemingly exists for some of the subspecies, although this could be a result of technical biases based on the limited number of plants tested, whereas some subspecies are not as stringent in their host range and can infect several plant hosts. Disease symptoms: Most X. fastidiosa‐related diseases appear as marginal leaf necrosis and scorching of the leaves. In the case of PD, X. fastidiosa can also cause desiccation of berries (termed ‘raisining’), irregular periderm development and abnormal abscission of petioles. In olive trees affected with OQDS, leaves exhibit marginal necrosis and defoliation, and overall tree decline occurs. Plants with ALS and OLS also exhibit the characteristic leaf scorch symptoms. Not all X. fastidiosa‐related diseases exhibit the typical leaf scorch symptoms. These include CVC and Phony Peach disease, amongst others. In the case of CVC, symptoms include foliar wilt and interveinal chlorosis on the upper surfaces of the leaves (similar to zinc deficiency), which correspond to necrotic, gum‐like regions on the undersides of the leaves. Additional symptoms of CVC include defoliation, dieback and hardening of fruits. Plants infected with Phony Peach disease exhibit a denser, more compact canopy (as a result of shortened internodes, darker green leaves and delayed leaf senescence), premature bloom and reduced fruit size. Some occlusions occur in the xylem vessels, but there are no foliar wilting, chlorosis or necrosis symptoms . Useful websites: http://www.piercesdisease.org/; https://pubmlst.org/xfastidiosa/; http://www.xylella.lncc.br/; https://nature.berkeley.edu/xylella/; https://ec.europa.eu/food/plant/plant_health_biosecurity/legislation/emergency_measures/xylella-fastidiosa_en [ABSTRACT FROM AUTHOR]

Summary: Pantoea agglomerans, a widespread epiphytic bacterium, has evolved into a hypersensitive response and pathogenicity (hrp)‐dependent and host‐specific gall‐forming pathogen by the acquisition of a pathogenicity plasmid containing a type III secretion system (T3SS) and its effectors (T3Es). Pantoea agglomerans pv. betae (Pab) elicits galls on beet (Beta vulgaris) and gypsophila (Gypsophila paniculata), whereas P. agglomerans pv. gypsophilae (Pag) incites galls on gypsophila and a hypersensitive response (HR) on beet. Draft genome sequences were generated and employed in combination with a machine‐learning approach and a translocation assay into beet roots to identify the pools of T3Es in the two pathovars. The genomes of the sequenced Pab4188 and Pag824‐1 strains have a similar size (∼5 MB) and GC content (∼55%). Mutational analysis revealed that, in Pab4188, eight T3Es (HsvB, HsvG, PseB, DspA/E, HopAY1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on beet and gypsophila. In Pag824‐1, nine T3Es (HsvG, HsvB, PthG, DspA/E, HopAY1, HopD1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on gypsophila, whereas the PthG effector triggers HR on beet. HsvB, HsvG, PthG and PseB appear to endow pathovar specificities to Pab and Pag, and no homologous T3Es were identified for these proteins in other phytopathogenic bacteria. Conversely, the remaining T3Es contribute to the virulence of both pathovars, and homologous T3Es were found in other phytopathogenic bacteria. Remarkably, HsvG and HsvB, which act as host‐specific transcription factors, displayed the largest contribution to disease development. [ABSTRACT FROM AUTHOR]

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis- trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl-coenzyme A (acyl-CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H2O2 and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild-type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1-only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease. [ABSTRACT FROM AUTHOR]

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild-type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence. [ABSTRACT FROM AUTHOR]

Citrus canker, caused by Xanthomonas citri, affects most commercial citrus varieties. All X. citri strains possess at least one transcription activator-like effector of the PthA family that activates host disease susceptibility ( S) genes. The X. citri strain 306 encodes four PthA effectors; nevertheless, only PthA4 is known to elicit cankers on citrus. As none of the PthAs act as avirulence factors on citrus, we hypothesized that PthAs 1-3 might also contribute to pathogenicity on certain hosts. Here, we show that, although PthA4 is indispensable for canker formation in six Brazilian citrus varieties, PthAs 1 and 3 contribute to canker development in 'Pera' sweet orange, but not in 'Tahiti' lemon. Deletions in two or more pthA genes reduce bacterial growth in planta more pronouncedly than single deletions, suggesting an additive role of PthAs in pathogenicity and bacterial fitness. The contribution of PthAs 1 and 3 in canker formation in 'Pera' plants does not correlate with the activation of the canker S gene, LOB1 ( LATERAL ORGAN BOUNDARIES 1), but with the induction of other PthA targets, including LOB2 and citrus dioxygenase ( DIOX). LOB1, LOB2 and DIOX show differential PthA-dependent expression between 'Pera' and 'Tahiti' plants that appears to be associated with nucleotide polymorphisms found at or near PthA-binding sites. We also present evidence that LOB1 activation alone is not sufficient to elicit cankers on citrus, and that DIOX acts as a canker S gene in 'Pera', but not 'Tahiti', plants. Our results suggest that the activation of multiple S genes, such as LOB1 and DIOX, is necessary for full canker development. [ABSTRACT FROM AUTHOR]

The mechanisms determining the host range of Xanthomonas are still undeciphered, despite much interest in their potential roles in the evolution and emergence of plant pathogenic bacteria. Xanthomonas citri pv. citri ( Xci) is an interesting model of host specialization because of its pathogenic variants: pathotype A strains infect a wide range of Rutaceous species, whereas pathotype A*/ AW strains have a host range restricted to Mexican lime ( Citrus aurantifolia) and alemow ( Citrus macrophylla). Based on a collection of 55 strains representative of Xci worldwide diversity assessed by amplified fragment length polymorphism ( AFLP), we investigated the distribution of type III effectors ( T3Es) in relation to host range. We examined the presence of 66 T3Es from xanthomonads in Xci and identified a repertoire of 28 effectors, 26 of which were shared by all Xci strains, whereas two ( xopAG and xopC1) were present only in some A*/ AW strains. We found that xopAG (= avrGf1) was present in all AW strains, but also in three A* strains genetically distant from AW, and that all xopAG-containing strains induced the hypersensitive response ( HR) on grapefruit and sweet orange. The analysis of xopAD and xopAG suggested horizontal transfer between X. citri pv. bilvae, another citrus pathogen , and some Xci strains. A strains were genetically less diverse, induced identical phenotypic responses and possessed indistinguishable T3E repertoires. Conversely, A*/ AW strains exhibited a wider genetic diversity in which clades correlated with geographical origin and T3E repertoire, but not with pathogenicity, according to T3E deletion experiments. Our data outline the importance of taking into account the heterogeneity of Xci A*/ AW strains when analysing the mechanisms of host specialization. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators ( RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide ( EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins. [ABSTRACT FROM AUTHOR]

Background Xanthomonas campestris pv. campestris ( Xcc) ( Pammel) Dowson is a Gram-negative bacterium that causes black rot, the most important disease of vegetable brassica crops worldwide. Intensive molecular investigation of Xcc is gaining momentum and several whole genome sequences are available. Taxonomy Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadacea; Genus Xanthomonas; Species X. campestris. Host range and symptoms Xcc can cause disease in a large number of species of Brassicaceae (ex- Cruciferae), including economically important vegetable Brassica crops and a number of other cruciferous crops, ornamentals and weeds, including the model plant Arabidopsis thaliana. Black rot is a systemic vascular disease. Typical disease symptoms include V-shaped yellow lesions starting from the leaf margins and blackening of the veins. Race structure, pathogenesis and epidemiology Collections of Xcc isolates have been differentiated into physiological races based on the response of several brassica species lines. Black rot is a seed-borne disease. The disease is favoured by warm, humid conditions and can spread rapidly from rain dispersal and irrigation water. Disease control The control of black rot is difficult and relies on the use of pathogen-free planting material and the elimination of other potential inoculum sources (infected crop debris and cruciferous weeds). Major gene resistance is very rare in B. oleracea (brassica C genome). Resistance is more readily available in other species, including potentially useful sources of broad-spectrum resistance in B. rapa and B. carinata ( A and BC genomes, respectively) and in the wild relative A. thaliana. Genome The reference genomes of three isolates have been released. The genome consists of a single chromosome of approximately 5 100 000 bp, with a GC content of approximately 65% and an average predicted number of coding DNA sequences ( CDS) of 4308. Important genes identified Three different secretion systems have been identified and studied in Xcc. The gene clusters xps and xcs encode a type II secretion system and xps genes have been linked to pathogenicity. The role of the type IV secretion system in pathogenicity is still uncertain. The hrp gene cluster encodes a type III secretion system that is associated with pathogenicity. An inventory of candidate effector genes has been assembled based on homology with known effectors. A range of other genes have been associated with virulence and pathogenicity, including the rpf, gum and wxc genes involved in the regulation of the synthesis of extracellular degrading enzymes, xanthan gum and lipopolysaccharides. Useful website http://www.xanthomonas.org/ [ABSTRACT FROM AUTHOR]

Xanthomonas citri ssp. citri ( Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm-deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn 5 transposon EZ- Tn 5 < KAN-2> Tnp Transposome ( Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ- Tn 5 was inserted at position 446 of hrp M, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrp M gene is involved in β-1,2-glucan synthesis in Xcc. A mutation in hrp M resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H2 O2 in comparison with the wild-type strain. All defective phenotypes were restored to wild-type levels by complementation with the plasmid p BBR1- MCS containing an intact copy of the hrp M gene and its promoter. These results indicate that the hrp M gene contributes to Xcc growth and adaptation in its host plant. [ABSTRACT FROM AUTHOR]

SUMMARY Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified. [ABSTRACT FROM AUTHOR]

SUMMARY Multilocus sequence analysis (MLSA) and type III effector (T3E) repertoire mining were performed to gain new insights into the genetic relatedness of Xanthomonas oryzae pv. oryzae ( Xoo) and Xanthomonas oryzae pv. oryzicola ( Xoc), two major bacterial pathogens of rice. Based on a collection of 45 African and Asian strains, we first sequenced and analysed three housekeeping genes by MLSA, Bayesian clustering and a median-joining network approach. Second, we investigated the distribution of 32 T3E genes, which are known to be major virulence factors of plant pathogenic bacteria, in all selected strains, by polymerase chain reaction and dot-blot hybridization methods. The diversity observed within housekeeping genes, as well as within T3E repertoires, clearly showed that both pathogens belong to closely related, but distinct, phylogenetic groups. Interestingly, these evolutionary groups are differentiated according to the geographical origin of the strains, suggesting that populations of Xoo and Xoc might be endemic in Africa and Asia, and thus have evolved separately. We further revealed that T3E gene repertoires of both pathogens comprise core and variable gene suites that probably have distinct roles in pathogenicity and different evolutionary histories. In this study, we carried out a functional analysis of xopO, a differential T3E gene between Xoo and Xoc, to determine the involvement of this gene in tissue specificity. Altogether, our data contribute to a better understanding of the evolutionary history of Xoo and Xoc in Africa and Asia, and provide clues for functional studies aiming to understand the virulence, host and tissue specificity of both rice pathogens. [ABSTRACT FROM AUTHOR]

SUMMARY One reason for the success of Pseudomonas syringae as a model pathogen has been the availability of three complete genome sequences since 2005. Now, at the beginning of 2011, more than 25 strains of P. syringae have been sequenced and many more will soon be released. To date, published analyses of P. syringae have been largely descriptive, focusing on catalogues of genetic differences among strains and between species. Numerous powerful statistical tools are now available that have yet to be applied to P. syringae genomic data for robust and quantitative reconstruction of evolutionary events. The aim of this review is to provide a snapshot of the current status of P. syringae genome sequence data resources, including very recent and unpublished studies, and thereby demonstrate the richness of resources available for this species. Furthermore, certain specific opportunities and challenges in making the best use of these data resources are highlighted. [ABSTRACT FROM AUTHOR]

SUMMARY [ABSTRACT FROM AUTHOR]

Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein–protein and protein–DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with α-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Δ ubc13 and Δ mms2/ uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair. [ABSTRACT FROM AUTHOR]

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour. [ABSTRACT FROM AUTHOR]

The RaxHR two-component regulatory system (TCS) of the rice pathogen Xanthomonas oryzae pv. oryzae is required for AvrXa21 activity. RaxH is a typical transmembrane histidine protein kinase (HK), whereas RaxR is its concomitant response regulator (RR). Here, we report the isolation of soluble, active amounts of recombinant His-tagged full-length RaxH and RaxR following growth of Escherichia coli over-expressing strains in the presence of sorbitol and glycine betaine. Full-length His-RaxH showed similar autophosphorylation activities to that of a truncated version of the protein (His-t-RaxH), lacking the N-terminal transmembrane region. Transphosphorylation assays revealed that only full-length RaxH was able to induce phosphorylation of His-RaxR, indicating that the N-terminal region of RaxH may be required for transphosphorylation of RaxR. Using site-directed mutagenesis we also demonstrated that residues histidine 222 in RaxH and aspartate 51 in RaxR are essential for phosphorylation activities of these proteins. Utilization of compatible solutes may be widely applied for purification of soluble, active recombinant transmembrane proteins, and in particular for purification of transmembrane HKs. [ABSTRACT FROM AUTHOR]

Leifsonia xyli ssp. x yli is the causative agent of ratoon stunting disease, a major cause of economic loss in sugarcane crops. Understanding of the biology of this pathogen has been hampered by its fastidious growth characteristics in vitro. However, the recent release of a genome sequence for this organism has allowed significant novel insights. Further to this, we have performed a bioinformatic analysis of the lipoproteins encoded in the L. xyli genome. These analyses suggest that lipoproteins represent c. 2.0% of the L. xyli predicted proteome. Functional analyses suggest that lipoproteins make an important contribution to the physiology of the pathogen and may influence its ability to cause disease in planta. [ABSTRACT FROM AUTHOR]

The DF signal molecule regulates the production of both yellow pigments (xanthomonadins) and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris. These two bacterial products are crucial to the epiphytic survival and pathogenicity of this pathogen on its plant hosts. Previous work suggested that DF is a butyrolactone, which the Streptomyces bacteria are known to utilize as signals. pigB is one of seven transcriptional units in the X. c. pv. campestris xanthomonadin gene cluster, and its inactivation results in the loss of DF signal, xanthomonadin and EPS production. Here, determination and analysis of the pigB DNA sequence reveals the presence of two open reading frames, the first ( xanB1) encoding a putative reductase/halogenase, and the second ( xanB2) showing the highest level of identity to Streptomyces genes encoding putative pteridine-dependent dioxygenase-like proteins. We show that xanB2 (but not xanB1) is needed for production of the DF signal, and that some Streptomyces strains produce functional analogues of DF. A role for xanB2 in the biosynthesis of DF is proposed. [ABSTRACT FROM AUTHOR]

The cruciferous weedArabidopsis thalianaand the causal agent of black rot disease of CrucifersXanthomonas campestrispv.campestris(Xcc) are both model organisms in plant pathology. Their interaction has been studied successfully in the past, but these investigations suffered from high variability. In the present study, we describe an improved Arabidopsis– Xccpathosystem that is based on a wound inoculation procedure. We show that after wound inoculation,Xcccolonizes the vascular system of Arabidopsis leaves and causes typical black rot symptoms in a compatible interaction, while in an incompatible interaction bacterial multiplication is inhibited. The highly synchronous and reproducible symptom expression allowed the development of a disease scoring scheme that enabled us to analyse the effects of mutations in individual genes on plant resistance or on bacterial virulence in a simple and precise manner. This optimized Arabidopsis– Xccpathosystem will be a robust tool for further genetic and post-genomic investigation of fundamental questions in plant pathology. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell-wall proteins that specifically inhibit fungal endo-polygalacturonases (PGs) that contribute to the aggressive decomposition of susceptible plant tissues. The inhibition of fungal PGs by PGIPs suggests that PGIPs have a role in plant tolerance to fungal infections and this has been observed in transgenic plants expressing PGIPs.Xylella fastidiosa, the causal agent of Pierce's disease (PD) in grapevines, has genes that encode cell-wall-degrading enzymes, including a putative PG. Therefore, we hypothesized that PGIP expression could confer tolerance against this bacterium as well as against the fungal pathogenBotrytis cinerea. To test this hypothesis,Vitis viniferacvs.‘Thompson Seedless’ and‘Chardonnay’ were transformed to express pear fruit PGIP-encoding gene (pPGIP) under the control of the CaMV 35S promoter. Substantial pear PGIP (pPGIP) activity was found in crude extracts from leaves and in xylem exudate of transgenic lines obtained from independent transformation events, but not in untransformed controls. pPGIP activity was detected in xylem exudate of untransformed scions grafted on to transgenic rootstocks expressingpPGIP. Leaves of transgenic plants infected withB. cinereahad reduced rates of lesion expansion. The development of PD was delayed in some transgenic lines with increased pPGIP activity. PD-tolerant transgenic lines had reduced leaf scorching, lowerXylellatitres and better re-growth after pruning than the untransformed controls. [ABSTRACT FROM AUTHOR]

Bacteria, Proteobacteria, gamma subdivision, Xanthomodales, Xanthomonas group, axonopodis DNA homology group, X. axonopodis pv. citri (Hasse) Vauterin et al . Gram negative, slender, rod-shaped, aerobic, motile by a single polar flagellum, produces slow growing, non-mucoid colonies in culture, ecologically obligate plant parasite. Causal agent of Asiatic citrus canker on most Citrus spp. and close relatives of Citrus in the family Rutaceae. Distinctively raised, necrotic lesions on fruits, stems and leaves. Bacteria exude from lesions during wet weather and are disseminated by splash dispersal at short range, windblown rain at medium to long range and human assisted movement at all ranges. Severe infections cause defoliation, blemished fruit, premature fruit drop, die-back of twigs and general debilitation of the tree. Citrus canker is not present in all subtropical to tropical regions of citriculture in the world, so considerable regulatory efforts are expended to prevent the introduction and spread of X. axonopodis pv. citri into areas in the Americas, Australia and elsewhere, with climates conducive to the disease. Limited strategies exist for suppression of citrus canker on more susceptible cultivars. Blemished fruit are unmarketable and exposed fruit are restricted in market access. The economic impact of loss of markets is much greater than that from yield and quality reductions of the crop. , , , , , , . [ABSTRACT FROM AUTHOR]

SUMMARY The Gram-negative, fastidious bacterium Xylella fastidiosa was successfully transformed with two RSF1010 derivative plasmids belonging to the incompatibility group IncQ, using electroporation. These two derivative plasmids, pXF004 and pXF005, were found to be present as autonomous, structurally unchanged DNA molecules when propagated in X. fastidiosa . However, neither pXF004 nor pXF005 were stably maintained in X. fastidiosa without antibiotic selection. When plasmid DNAs were isolated from X. fastidiosa , or plasmid DNAs isolated from E. coli were supplemented with a TypeOne™ inhibitor, TRI, the frequency of transformation was increased by 13- or 5-fold, respectively. Plasmid pXF005 was also used to transform one additional grapevine strain of X. fastidiosa. [ABSTRACT FROM AUTHOR]

Taxonomy: Bacteria; Proteobacteria, Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae, Xanthomonas. Microbiological properties: Gram-negative, obligately aerobic, straight rods, motile by a single polar flagellum, yellow pigment. Related species: X. campestris,X. axonopodis,X. oryzae,X. albilineans. Host range: Affects Rutaceous plants, primarily Citrus spp., Fortunella spp., and Poncirus spp., world-wide. Quarantined pathogen in many countries. Economically important hosts are cultivated orange, grapefruit, lime, lemon, pomelo and citrus rootstock. Disease symptoms: On leaves, first appearance is as oily looking, 2-10 mm, similarly sized, circular spots, usually on the abaxial surface. On leaves, stems, thorns and fruit, circular lesions become raised and blister-like, growing into white or yellow spongy pustules. These pustules then darken and thicken into a light tan to brown corky canker, which is rough to the touch. On stems, pustules may coalesce to split the epidermis along the stem length, and occasionally girdling of young stems may occur. Older lesions on leaves and fruit tend to have more elevated margins and are at times surrounded by a yellow chlorotic halo (that may disappear) and a sunken centre. Sunken craters are especially noticeable on fruit, but the lesions do not penetrate far into the rind. Defoliation and premature abscission of affected fruit occurs on heavily infected trees. [ABSTRACT FROM AUTHOR]