Tritrophic interactions among Macrosiphum euphorbiae aphids, their host plants and endosymbionts: Investigation by a proteomic approach

The Mi-1.2 gene in tomato confers resistance against certain clones of the potato aphid (Macrosiphum euphorbiae). This study used 2D-DIGE coupled with protein identification by MALDI-TOF-MS to compare the proteome patterns of avirulent and semivirulent potato aphids and their bacterial endosymbionts on resistant (Mi-1.2+) and susceptible (Mi-1.2−) tomato lines. Avirulent aphids had low survival on resistant plants, whereas the semivirulent clone could colonize these plants. Eighty-two protein spots showed significant quantitative differences among the four treatment groups, and of these, 48 could be assigned putative identities. Numerous structural proteins and enzymes associated with primary metabolism were more abundant in the semivirulent than in the avirulent aphid clone. Several proteins were also up-regulated in semivirulent aphids when they were transferred from susceptible to resistant plants. Nearly 25% of the differentially regulated proteins originated from aphid endosymbionts and not the aphid itself. Six were assigned to the primary endosymbiont Buchnera aphidicola, and 5 appeared to be derived from a Rickettsia-like secondary symbiont. These results indicate that symbiont expression patterns differ between aphid clones with differing levels of virulence, and are influenced by the aphids’ host plant. Potentially, symbionts may contribute to differential adaptation of aphids to host plant resistance.     

Comparative proteomic analysis of bacterial wilt susceptible and resistant tomato cultivars

To investigate the molecular mechanisms of bacterial resistance in susceptible and resistant cultivars of tomato, a proteomic approach was adopted. Four cultivars of tomato were selected on the basis of their response to bacterial (Pseudomonas solanacearum) inoculation wherein cultivar Roma and Riogarande, and cultivar Pusa Ruby and Pant Bahr were considered as resistant and susceptible cultivars, respectively. Proteins were extracted from leaves of 3-week-old seedlings of the four cultivars and separated by 2-DE. A total of nine proteins were found to be differentially expressed between the susceptible and resistant cultivars. Amino acid sequences of these proteins were determined with a protein sequencer. The identified proteins belongs to the categories of energy, protein destination and storage, and defense. Of these proteins, a 60 kDa chaperonin and an apical membrane antigen were significantly upregulated in resistant cultivars compared with susceptible cultivars. Application of jasmonic acid and salicylic acid resulted in significant changes in levels of apical membrane antigen and protein disulfide-isomerase. Taken together, these results suggest that apical membrane antigen might be involved in bacterial resistance process through salicylic acid induced defense mechanism signaling in tomato plants.     

Constitutive and Inducible Proteins in the Root of Chinese Cabbage Infected with Plasmodiophora brassicae

A plant-pathogen system consisting of a Chinese cabbage cultivar and two isolates of Plasmodiophora brassicae was developed for analysing root proteins accumulated in susceptible and resistant responses to the fungus. Proteins extracted at pH 2.8 were analysed by two-dimensional gel electrophoresis. More than 150 protein spots were resolved. Spots indicating changes in the intensity by the infection of P. brassicae were classified into six types: class 1 contains proteins enhanced in susceptible response; class 2, proteins unique to susceptible response; class 3, proteins repressed in susceptible response: class 4, proteins enhanced in resistant response; class 5, proteins unique to resistant response; and class 6, proteins repressed in resistant response. Two proteins from class 1 and one protein from class 4 were subjected to an N-terminal amino acid sequencing. One of the class 1 protein (25 kDa, pl 7.0) revealed high homology with pathogenesis-related protein group 5.     

Investigation of the effect of nitrogen on severity of Fusarium Head Blight in barley

The effect of nitrogen on Fusarium Head Blight (FHB) in a susceptible barley cultivar was investigated using gel-based proteomics. Barley grown with either 15 or 100 kg ha^? 1 N fertilizer was inoculated with Fusarium graminearum (Fg). The storage protein fraction did not change significantly in response either to N level or Fg, whereas eighty protein spots in the water-soluble albumin fraction increased and 108 spots decreased more than two-fold in intensity in response to Fg. Spots with greater intensity in infected plants contained fungal proteins (9 spots) and proteolytic fragments of plant proteins (65 spots). Identified fungal proteins included two superoxide dismutases, l-xylulose reductase in two spots, peptidyl prolyl cis–trans isomerase and triosephosphate isomerase, and proteins of unknown function. Spots decreasing in intensity in response to Fg contained plant proteins possibly degraded by fungal proteases. Greater spot volume changes occurred in response to Fg in plants grown with low nitrogen, although proteomes of uninfected plants were similar for both treatments. Correlation of proteome changes with measurement of Fusarium-damaged kernels, fungal biomass and mycotoxin levels indicated that increased Fusarium infection occurred in barley with low N and suggests control of N fertilization as a possible way to minimise FHB in barley.     

Characterization and evaluation of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strain WF02 regarding its biocontrol activities and genetic responses against bacterial wilt in two different resistant tomato cultivars

Bacillus amyloliquefaciens strain WF02, isolated from soil collected at Wufeng Mountain, Taiwan, has siderophore-producing ability and in vitro antagonistic activity against bacterial wilt pathogen. To determine the impact of plant genotype on biocontrol effectiveness, we treated soil with this strain before infecting susceptible (L390) and moderately resistant (Micro-Tom) tomato cultivars with Ralstonia solanacearum strain Pss4. We also compared the efficacy of this strain with that of commercial Bacillus subtilis strain Y1336. Strain WF02 provided longer lasting protection against R. solanacearum than did strain Y1336 and controlled the development of wilt in both cultivars. To elucidate the genetic responses in these plants under WF02 treatment, we analyzed the temporal expression of defense-related genes in leaves. The salicylic acid pathway-related genes phenylalanine ammonia-lyase and pathogenesis-related protein 1a were up-regulated in both cultivars, whereas expression of the jasmonic acid pathway-related gene lipoxygenase was only elevated in the susceptible tomato cultivar (L390). These results suggest that WF02 can provide protection against bacterial wilt in tomato cultivars with different levels of disease resistance via direct and indirect modes of action.     

The Viable But Nonculturable State of Ralstonia solanacearum May Be Involved in Long-Term Survival and Plant Infection

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.     

Serological Screening of the Schistosoma mansoni Adult Worm Proteome

Background

New interventions tools are a priority for schistosomiasis control and elimination, as the disease is still highly prevalent. The identification of proteins associated with active infection and protective immune response may constitute the basis for the development of a successful vaccine and could also indicate new diagnostic candidates. In this context, post-genomic technologies have been progressing, resulting in a more rational discovery of new biomarkers of resistance and antigens for diagnosis.

Methodology/Principal Findings

Two-dimensional electrophoresed Schistosoma mansoni adult worm protein extracts were probed with pooled sera of infected and non-infected (naturally resistant) individuals from a S. mansoni endemic area. A total of 47 different immunoreactive proteins were identified by mass spectrometry. Although the different pooled sera shared most of the immunoreactive protein spots, nine protein spots reacted exclusively with the serum pool of infected individuals, which correspond to annexin, major egg antigen, troponin T, filamin, disulphide-isomerase ER-60 precursor, actin and reticulocalbin. One protein spot, corresponding to eukaryotic translation elongation factor, reacted exclusively with the pooled sera of non-infected individuals living in the endemic area. Western blotting of two selected recombinant proteins, major egg antigen and hemoglobinase, showed a similar recognition pattern of that of the native protein.

Concluding/Significance

Using a serological proteome analysis, a group of antigens related to the different infection status of the endemic area residents was identified and may be related to susceptibility or resistance to infection.     

The influence of the fungal pathogen Mycocentrospora acerina on the proteome and polyacetylenes and 6-methoxymellein in organic and conventionally cultivated carrots (Daucus carota) during post harvest storage

Many carrots are discarded during post harvest cold storage due to development of fungal infections, caused by, e.g., Mycocentrospora acerina (liquorice rot). We compared the susceptibility of carrots grown under conventional and organic agricultural practices. In one year, organically cultivated carrots showed 3 × to 7 × more symptoms than conventionally cultivated, when studying naturally occurring disease at 4 and 6 months, respectively. On the other hand, we have developed a bioassay for infection studies of M. acerina on carrots and observed that organic roots were more susceptible after one month of storage than conventional ones, but no differences were apparent after four or six months storage. Levels of polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) did not change, whereas the isocoumarin phytoalexin (6-methoxymellein) accumulated in infected tissue as well as in healthy tissue opposite the infection. The proteomes of carrot and M. acerina were characterized, the intensity of 33 plant protein spots was significantly changed in infected roots including up regulation of defence and stress response proteins but also a decrease of proteins involved in energy metabolism. This combined metabolic and proteomic study indicates that roots respond to fungal infection through altered metabolism: simultaneous induction of 6-methoxymellein and synthesis of defence related proteins.     

Effect of temperature,cultivars, injury of root and inoculums load of Ralstonia solanacearum to cause bacterial wilt of tomato

Bacterial wilt, caused by Ralstonia solanacearum , is responsible for severe losses in tomato crops in the world. In the present study, the effect of temperature, cultivars of tomato, injury of root system and inoculums load of R. solanacearum to cause bacterial wilt disease under control conditions was undertaken. Three strains UTT-25, HPT-3 and JHT-5 of R. solanacearum were grown at 5–40?°C in vitro to study, the effect of temperature on the growth of bacteria and maximum growth was found at 30?°C after 72?h in all the strains. Twenty-one days old seedlings of two cultivars of tomato i.e. N-5 (moderately resistant) and Pusa Ruby (highly susceptible) were transplanted into the pots and inoculated with R. solanacearum strain UTT-25 (5 × 10⁸?cfu/ml), mechanically injured and uninjured roots of the plant. The plants were allowed to grow at 20, 25, 30 and 35?°C at National Phytotron Facility, IARI, New Delhi to study the effect of temperature on intensity of bacterial wilt disease. Maximum wilt disease intensity was found 98.73 and 95.9 % in injured roots of Pusa Ruby and N-5 cultivars of tomato at 35?°C on 11th days of inoculation, respectively. However, no wilt disease was observed in both the cultivars at 20?°C up to 60?days. For detection of R. solanacearum from asymptomatic tomato plants, hrpB-based sequence primers (Hrp_rs2F and Hrp_rs2R) amplified at 323?bp was used in bio-PCR to detect R. solanacearum from crown, mid part of stem and upper parts of the plant. Another experiment was conducted to find out the inoculum potential of R. solanacearum strain UTT-25 to cause bacterial wilt in susceptible cultivar Pusa Ruby. The bacteria were inoculated at concentration of bacterial suspension 10 to 10¹⁰?cfu/ml in injured and uninjured roots of the plants separately and injured root accelerated wilt incidence and able to cause wilt disease 63.3% by 100?cfu/ml of R. solanacearum, while no disease appeared at 10?cfu/ml on the 11th day of inoculation in injured and uninjured roots of the plant.     

Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

The Ralstonia solanacearum csp22 peptide,but not flagellin‐derived peptides,is perceived by plants from the Solanaceae family

Ralstonia solanacearum, the causal agent of bacterial wilt disease, is considered one of the most destructive bacterial pathogens due to its lethality, unusually wide host range, persistence and broad geographical distribution. In spite of the extensive research on plant immunity over the last years, the perception of molecular patterns from R. solanacearum that activate immunity in plants is still poorly understood, which hinders the development of strategies to generate resistance against bacterial wilt disease. The perception of a conserved peptide of bacterial flagellin, flg22, is regarded as paradigm of plant perception of invading bacteria; however, no elicitor activity has been detected for R. solanacearum flg22. Recent reports have shown that other epitopes from flagellin are able to elicit immune responses in specific species from the Solanaceae family, yet our results show that these plants do not perceive any epitope from R. solanacearum flagellin. Searching for elicitor peptides from R. solanacearum, we found several protein sequences similar to the consensus of the elicitor peptide csp22, reported to elicit immunity in specific Solanaceae plants. A R. solanacearum csp22 peptide (csp22^Rsol) was indeed able to trigger immune responses in Nicotiana benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22^Rsol treatment conferred increased resistance to R. solanacearum in tomato. Transgenic A. thaliana plants expressing the tomato csp22 receptor (SlCORE) gained the ability to respond to csp22^Rsol and became more resistant to R. solanacearum infection. Our results shed light on the mechanisms for perception of R. solanacearum by plants, paving the way for improving current approaches to generate resistance against R. solanacearum.     

Are Pathogenesis-Related Proteins Induced by Meloidogne javanica or Heterodera avenae lnvasion?

Changes in root- and leaf-soluble proteins were investigated in tomato after invasion by the root-knot nematode Meloidogyne javanica, or in barley and wheat after invasion by the cereal cyst nematode Heterodera avenae. Infection of susceptible tomato plants by M. javanica did not cause any change in the soluble-protein composition of leaves or roots compared with uninoculated plants at an early infection stage. No pathogenesis-related proteins (chitinase, glucanase, or P-14) were induced in the leaf apoplast. Changes in leaf proteins were not observed after invasion of wheat cultivars by H. avenae, whereas, in barley, a few changes in intercellular leaf proteins were recorded in resistant cultivars. These changes, however, were not the same among different H. avenae-resistant cultivars. Protein changes were found at an early stage of infection in barley and wheat roots infected with H. avenae, but no difference was found between resistant and susceptible cultivars.     

Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector,Ixodes scapularis

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.