Angela Mehta

Plant–pathogen interactions: what is proteomics telling us?

Over the years, several studies have been performed to analyse plant–pathogen interactions. Recently, functional genomic strategies, including proteomics and transcriptomics, have contributed to the effort of defining gene and protein function and expression profiles. Using these ‘omic’ approaches, pathogenicity‐ and defence‐related genes and proteins expressed during phytopathogen infections have been identified and enormous datasets have been accumulated. However, the understanding of molecular plant–pathogen interactions is still an intriguing area of investigation. Proteomics has dramatically evolved in the pursuit of large‐scale functional assignment of candidate proteins and, by using this approach, several proteins expressed during phytopathogenic interactions have been identified. In this review, we highlight the proteins expressed during plant–virus, plant–bacterium, plant–fungus and plant–nematode interactions reported in proteomic studies, and discuss these findings considering the advantages and limitations of current proteomic tools.

Phylogenetic relationships of Xylella fastidiosa strains from different hosts, based on 16S rDNA and 16S-23S intergenic spacer sequences.

The phylogenetic relationships of Xylella fastidiosa strains isolated from different hosts, including citrus trees, coffee, grapevine, plum and pear, were inferred by sequence analysis of the 16S rDNA and 16S-23S intergenic spacer region. A high level of similarity (97.1-100%) was found in the 16S rDNA of the Xylella fastidiosa strains. The 16S-23S region showed a higher level of variation, with similarity values ranging from 79.8 to 100%. Two tRNAs (tRNA(Ala) and tRNA(Ile)) were encountered within the spacer sequence. The phylogenetic trees, constructed using the neighbour-joining method, showed that the citrus, coffee, peach and plum strains were closely related and separate from grapevine strains. The pear strain remained isolated from all the other Xylella strains in both analyses and produced values of less than 20% in DNA-DNA hybridization experiments with a citrus strain. These results show that this strain does not belong to the Xylella fastidiosa genomic species.

A simple method for in vivo expression studies of Xanthomonas axonopodis pv. citri.

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.

In vivo proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the host plant Brassica oleracea

The genus Xanthomonas is composed of several species that cause severe crop losses around the world. In Latin America, one of the most relevant species is Xanthomonas campestris pv. campestris, which is responsible for black rot in cruciferous plants. This pathogen causes yield losses in several cultures, including cabbage, cauliflower and broccoli. Although the complete structural genome of X. campestris pv. campestris has been elucidated, little is known about the protein expression of this pathogen in close interaction with the host plant. Recently, a method for in vivo analysis of Xanthomonas axonopodis pv. citri was developed. In the present study, this technique was employed for the characterization of the protein expression of X. campestris pv. campestris in close interaction with the host plant Brassica oleracea. The bacterium was infiltrated into leaves of the susceptible cultivar and later recovered for proteome analysis. Recovered cells were used for protein extraction and separated by two-dimensional electrophoresis. Proteins were analysed by peptide mass fingerprinting or de novo sequencing and identified by searches in public databases. The approach used in this study may be extremely useful in further analyses in order to develop novel strategies to control this important plant pathogen.

Rooteomics: The Challenge of Discovering Plant Defense-Related Proteins in Roots

In recent years, a strong emphasis has been given in deciphering the function of genes unraveled by the completion of several genome sequencing projects. In plants, functional genomics has been massively used in order to search for gene products of agronomic relevance. As far as root-pathogen interactions are concerned, several genes are recognized to provide tolerance/resistance against potential invaders. However, very few proteins have been identified by using current proteomic approaches. One of the major drawbacks for the successful analysis of root proteomes is the inherent characteristics of this tissue, which include low volume content and high concentration of interfering substances such as pigments and phenolic compounds. The proteome analysis of plant-pathogen interactions provides important information about the global proteins expressed in roots in response to biotic stresses. Moreover, several pathogenic proteins superimpose the plant proteome and can be identified and used as targets for the control of viruses, bacteria, fungi and nematode pathogens. The present review focuses on advances in different proteomic strategies dedicated to the challenging analysis of plant defense proteins expressed during bacteria-, fungi- and nematode-root interactions. Recent developments, limitations of the current techniques, and technological perspectives for root proteomics aiming at the identification of resistance-related proteins are discussed.

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination

BackgroundAsian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris.ResultsA cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%.ConclusionsOur data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

Proteomic analysis of Metarhizium anisopliae secretion in the presence of the insect pest Callosobruchus maculatus.

Crop improvement in agriculture generally focuses on yield, seed quality and nutritional characteristics, as opposed to resistance to biotic stresses. Consequently, natural antifeedant toxins are often rare in seed material, with commercial crops being prone to insect pest predation. In the specific case of cowpea (Vigna unguiculata), smallholder cropping is affected by insect pests that reproduce inside the stored seeds. Entomopathogenic organisms can offer an alternative to conventional pesticides for pest control, producing hydrolases that degrade insect exoskeleton. In this study, protein secretions of the ascomycete Metarhizium anisopliae, which conferred bioinsecticidal activity against Callosobruchus maculatus, were characterized via 2D electrophoresis and mass spectrometry. Proteases, reductases and acetyltransferase enzymes were detected. These may be involved in degradation and nutrient uptake from dehydrated C. maculatus. Proteins identified in this work allowed description of metabolic pathways. Their potential applications in biotechnology include both novel compound development and production of genetically modified plants resistant to insect pests.

Comparative proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the susceptible and the resistant cultivars of Brassica oleracea

Black rot of cruciferous plants, caused by Xanthomonas campestris pv. campestris, causes severe losses in agriculture around the world. This disease affects several cultures, including cabbage and broccoli, among others. Proteome studies of this bacterium have been reported; however, most of them were performed using the bacterium grown under culture media conditions. Recently, we have analyzed the proteome of X. campestris pv. campestris during the interaction with the susceptible cultivar of Brassica oleracea and several proteins were identified. The objective of the present study was to analyze the expressed proteins of X. campestris pv. campestris during the interaction with the resistant cultivar of B. oleracea. The bacterium was infiltrated in the leaves of the resistant plant and recovered for protein extraction and two-dimensional electrophoresis. The protein profile was compared with that of the bacterium isolated from the susceptible host and the results obtained revealed a group of proteins exclusive to the resistant interaction. Among the proteins identified in this study were plant and bacterium proteins, some of which were exclusively expressed during the resistant interaction.

Diversity analysis of Bemisia tabaci biotypes: RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region

The Bemisia tabaci complex is formed by approximately 41 biotypes, two of which (B and BR) occur in Brazil. In this work we aimed at obtaining genetic markers to assess the genetic diversity of the different biotypes. In order to do that we analyzed Bemisia tabaci biotypes B, BR, Q and Cassava using molecular techniques including RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region. The analyses revealed a high similarity between the individuals of the B and Q biotypes, which could be distinguished from the BR individuals. A phylogenetic tree based on ITS1 rDNA sequence was constructed. This is the first report of the ITS1 rDNA sequence of Bemisia tuberculata and of the BR biotype of B. tabaci.

Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.).

UNLABELLED In the present study we have identified and characterized the proteins expressed during different developmental stages of Elaeis guineensis calli obtained from zygotic embryos. We were interested in the possible proteomic changes that would occur during the acquisition of somatic embryogenesis and therefore samples were collected from zygotic embryos (E1), swollen explants 14days (E2) in induction medium, primary callus (E3), and pro-embryogenic callus (E4). The samples were grinded in liquid nitrogen, followed by total protein extraction using phenol and extraction buffer. Proteins were analyzed by two-dimensional electrophoresis (2-DE) and the differentially expressed protein spots were analyzed by MALDI-TOF mass spectrometry (MS and MS/MS). Interestingly, we have identified proteins, which can be used as potential candidates for future studies aiming at the development of biomarkers for embryogenesis acquisition and for the different stages leading to pro-embryogenic callus formation such as type IIIa membrane protein cp-wap13, fructokinase and PR proteins. The results obtained shed some light on the biochemical events involved in the process of somatic embryogenesis of E. guineensis obtained from zygotic embryos. The use of stage-specific protein markers can help monitor cell differentiation and contribute to improve the protocols for successfully cloning the species. BIOLOGICAL SIGNIFICANCE Understanding the fate and dynamics of cells and tissues during callus formation is essential to understand totipotency and the mechanisms involved during acquisition of somatic embryogenesis (SE). In this study we have investigated the early stages of somatic embryogenesis induction in oil palm and have identified potential markers as well as proteins potentially involved in embryogenic competence acquisition. The use of these proteins can help improve tissue culture protocols in order to increase regeneration rates. This article is part of a Special Issue entitled: Environmental and structural proteomics.