Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris

RpfG is a paradigm for a class of widespread bacterial two-component regulators with a CheY-like receiver domain attached to a histidine-aspartic acid-glycine-tyrosine-proline (HD-GYP) cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), a two-component system comprising RpfG and the complex sensor kinase RpfC is implicated in sensing and responding to the diffusible signaling factor (DSF), which is essential for cell-cell signaling. RpfF is involved in synthesizing DSF, and mutations of rpfF, rpfG, or rpfC lead to a coordinate reduction in the synthesis of virulence factors such as extracellular enzymes, biofilm structure, and motility. Using yeast two-hybrid analysis and fluorescence resonance energy transfer experiments in Xcc, we show that the physical interaction of RpfG with two proteins with diguanylate cyclase (GGDEF) domains controls a subset of RpfG-regulated virulence functions. RpfG interactions were abolished by alanine substitutions of the three residues of the conserved GYP motif in the HD-GYP domain. Changing the GYP motif or deletion of the two GGDEF-domain proteins reduced Xcc motility but not the synthesis of extracellular enzymes or biofilm formation. RpfG-GGDEF interactions are dynamic and depend on DSF signaling, being reduced in the rpfF mutant but restored by DSF addition. The results are consistent with a model in which DSF signal transduction controlling motility depends on a highly regulated, dynamic interaction of proteins that influence the localized expression of cyclic di-GMP.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected rootstock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Downregulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Determination of polyamines in organic extracts from roots of Canavalia ensiformis by capillary electrophoresis

A rapid, selective and specific capillary zone electrophoresis method to determine polyamines in organic extracts from roots of Canavalia ensiformis (Jack Beans) was developed using ultra violet (UV) detection. Canavalia ensiformis is relatively free from diseases and it is used as reference in allelopathy studies. Polyamines are widely distributed in plant and it could be involved in plant pathogen interactions. Optimal separation was achieved using 15 mmol.L-1formic acid (pH 3.0) + 4 mmol.L-1 imidazole as a background electrolyte. It was possible to identify and quantify the polyamines on herbal samples in the presence of other phytochemical substances and analyze them quickly (up to 6 min). The applicability of this method was evaluated in crude organic extracts from roots of Canavalia ensiformis.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

Investigation of plasmodiophora brassicae (clubroot disease) in vegetable brassica using arabidopsis thaliana as a model system

Clubroot, caused by Plasmodiophora brassicae, is the most devastating soil-borne disease of vegetable brassicas. It occurs all over the world and is responsible for crop losses of up to 10% every year. In Australia, the disease is being managed effectively with chemicals and cultural practices, but ideally control can be improved in the long term by the introduction of resistant cultivars. The life cycle ofP. brassicae and mode of action of plant resistance has not been fully elucidated because of the technical difficulties of working with an obligate, soil-borne plant pathogen. However, Arabidopsis thaliana, which is a host ofP. brassicae, has great potential as a model system for studying the life cycle, the infection process and development of resistance. We have developed a sand-liquid-culture system for growing Arabidopsis that allows easy observation of all life stages and, most importantly, the primary plasmodial stages within the root hair. The method was first optimised for observations of the lifecycle of the pathogen in a susceptible Arabidopsis ecotype (Col-3) where all stages of the lifecycle have now been observed and characterised. Further screening of Arabidopsis ecotypes for disease resistance has utilised one of the most virulent Australian pathotypes of brassica (ECD number 16/19/31). To date, Arabidopsis ecotype Ta-0 has shown a level of tolerance to the disease even though the roots get infected. It has been reported earlier that resistance toP. brassicae in Arabidopsis is due to one or a small number of genes. To examine changes in gene expression during the early, critical stages of infection, RNA was extracted from the susceptible and resistant ecotypes at two time points, 4 days and 17 days after inoculation. Microarray analysis will be used to investigate genome wide changes in gene expression during infection but also to identify candidate genes that may confer resistance to Australian isolates of the pathogen.

Use of high resolution digital multi-spectral imagery to assess the distribution of disease caused by Phytophthora cinnamomi on heathland at Anglesea, Victoria

Disease caused by the soilborne plant pathogen Phytophthora cinnamomi causes long-term floristic and structural changes in native vegetation communities in Australia. Key components of the management of this disease are to know where it occurs and the rate at which it spreads. The distribution of P. cinnamomi has generally been assessed as locality points of infestation and mapping the extent of diseased vegetation in any area is difficult and costly. This study was undertaken in P. cinnamomi-infested heathland communities in southern Victoria, Australia, where the symptoms of P. cinnamomi arise as a mosaic within healthy vegetation. We investigated the potential to improve the efficiency and effectiveness of mapping and monitoring vegetation affected by P. cinnamomi using digital multi-spectral imaging. This technique was developed for the purposes of monitoring vegetation and provides a single, seamless ortho-rectified digital image over the total area of interest. It is used to spatially quantify small differences in the characteristics of vegetation. In this study, the symptoms of disease caused by P. cinnamomi infestation were related to differences in the imagery and were used to map areas of infestation. Comparison of the digital multi-spectral imaging indications with on-ground observations gave moderate accuracy between the datasets (κ = 0.49) for disease and healthy indications. This study demonstrates the ability of the technique to determine disease extent over broad areas in native vegetation and provides a non-invasive, cost effective tool for management.

Effects of vegetation, fire and other disturbance factors on small mammal ecology and conservation

The relationship of vegetation and disturbance factors to the distribution, abundance and diversity of small mammals in the eastern Otway region, Victoria were investigated. Antechinus stuartii, Rattus fuscipes and Rattus lutreolus were widely distributed and occurred in the majority of the eleven floristic vegetation groups identified. Antechinus minimus, Antechinus swainsonnii and Pseudomys novaehollandiae had restricted distributions and were recorded in only two or three vegetation groups. New information on the distribution of the rare species P. novaehollandiae, was obtained and two floristically rich vegetation groups that it preferred were identified. Species-rich small mammal communities occurred in vegetation communities with high numbers of sclerophyll plant species and high structural diversity. Maximum food resources were considered to be provided in these communities. Local habitat diversity was also correlated with species-richness. Small mammal abundance was maximum in non-sclerophyllous canmunities, where high plant productivity was considered to be important. For the first time, the presence of the plant pathogen Phytophthora cinnamomi was shown to affect small mammals. It was associated with small mammal communities of low species richness and abundance, Recovery of small mammal populations after wildfire was slow until the fourth year. Mus musculus reached peak abundance from 2-3 years and then declined rapidly. P. novaehollandiae was the only native species that achieved maximum abundance early in the succession. A. stuartii, R. fuscipes and R. lutreolus approached maximum abundance in mid-succession, while Isoodon obesulus was a mid- to late-successional species. A. minimus survived the fire, but did not persist after one year. The pattern of succession was influenced by attributes of species, such as survival after fire, their ability to disperse and reproduce.

Effects of phytophthora cinnamomi on heathland flora and fauna of the Eastern Otway Ranges

The plant pathogen, Phytophthora dnnamomi, is a cause of dieback disease observed in sclerophyll vegetation in Australia, The effects of P. dnnamomi on flora and fauna were studied at two locations in heathland vegetation near the coastal town of Anglesea, Victoria. The pathogen was isolated from soils beneath diseased heathland plants. The extent of diseased vegetation was assessed by the presence and absence of highly sensitive indicator species, Xanthorrhoea australis and hopogon ceratophyllus. The characteristics of heathland vegetation exhibiting dieback disease associated with the presence of P. dnnamomi were investigated. Plant species richness was similar between diseased and non-diseased areas however diseased areas were characterised by significant declines in the cover and frequency of susceptible species, increases in resistant species and increases in percent cover of open ground. Compared to non-diseased areas, diseased areas exhibited fewer shrub species and decreased shrub cover. The percentage cover and number of species of sedges, lilies and grasses were higher in diseased areas. Structural differences were significant between 0-0.6 m with decreased cover of vegetation in diseased areas. Differences in structure between diseased and non-diseased areas were not as great as expected due to increases in the cover of resistant species. A number of regenerating X australis were observed in post-disease areas. Cluster analysis of floristic data could clearly separate diseased and non-diseased trap stations. The population dynamics and habitat use of eight small mammal species present were compared in diseased and non-diseased areas using trapping and radio-tracking techniques. The number of small mammal species captured in post-disease areas was significantly lower than non-diseased areas. Mean captures of Antechinus stuartii and Rattus fiisdpes were significantly lower in diseased areas on Grid B. Mean captures of Rattus lutreolus were significantly lower in diseased areas on both study grids. Significant differences were not observed in every season over the two year study period. Radio tracking revealed more observations of Sminthopsis leucopus in non-diseased vegetation than in diseased. Cercartetus nanus was frequently observed to utilise the disease susceptible X. australis for nesting. At one location, the recovery of vegetation and small mammal communities in non-diseased and diseased vegetation after fuel reduction burning was monitored for three years post-fire. Return of plant species after fire in both disease classes were similar, reaching 75% of pre-fire richness after three years. Vegetation cover was slower to return after fire in diseased areas. Of the seven small mammal species captured pre-fire, five were regularly captured in the three years after fire. General linear model analysis revealed a significant influence of disease on capture rates for total small mammals before fire and a significant influence of fire on capture rates for total small mammals after fire. After three years, the influence of fire on capture rates was reduced no significant difference was detected between disease classes. Measurements of microclimate indicate that diseased, burnt heathland was likely to experience greater extremes of temperature and wind speed. Seeding of diseased heathland with X. australis resulted in the establishment of seedlings of this sensitive species. The reported distributions of the mamma] species in Victoria were analysed to determine which species were associated with the reported distribution of dieback disease. Twenty-two species have more than 20% of their known distribution in diseased areas. Five of these species, Pseudomys novaehollandiae, Pseudomys fumeust Pseudomys shortridgei, Potorous longipes and Petrogale pencillata are rare or endangered in Victoria. Four of the twenty-two species, Sminthopsis leucopus, Isoodon obesulus, Cercartetus nanus and Rottus lutreolus am observed in Victorian heathlands. Phytophthora cinnamomi changes both the structure and floristics of heathland vegetation in the eastern Qtway Ranges. Small mammals respond to these changes through decreased utilisation of diseased heathland. The pathogen threatens the diversity of species present and future research efforts should be directed towards limiting its spread and rehabilitating diseased areas.

Variation in Cyclaneusma minus isolates

Cyclaneusma minus is a plant pathogen that has financial impacts on the forestry industry. This study characterised the genetic variation in Australian and New Zealand isolates of Cyclaneusma minus using both molecular and morphological techniques and developed a PCR detection test for the presence of Cyclaneusma minus in tree plantations.

Effects of Phytophthora cinnamomi on heathland fauna and ecosystem function

Progression of disease caused by the plant pathogen Phytophthora cinnamomi was correlated to rainfall events and resulted in a loss of plant species diversity in heathland vegetation at Anglesea, Victoria. Lower captures of small mammals were recorded in diseased areas. Management of disease using the chemical phosphite was also evaluated.

Responses of Arabidopsis thaliana to UV-C radiation and Hyaloperonospora arabidopsidis

 This work demonstrates the relationship between two seemingly unrelated stresses, and shows that exposing plants to UV radiation can increase their resistance to disease caused by a plant pathogen.

Controle biológico de Colletotrichum acutatum, agente causal da queda prematura dos frutos cítricos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in 'Valencia' oranges

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise do perfil de expressão dos genes da cana-de-açúcar envolvidos na interação com Leifsonia xyli subsp: xyli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

New genes of Xanthomonas citri subsp citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)