Current Proteomics - Volume 7, Issue 4, 2010

Since the early days of the human history, the development of agriculture has been crucial for the first human settlements and its transformation in our first cities. Nowadays, agriculture is still one of the main human activities. The development of molecular tools has increased the amount of relevant biological information directed to improve crops maintenance in several ways, such as plant development or its resistance to plant diseases. Between these molecular techniques, proteomics tools and technologies have become a great relevance during the last years. Some authors have labeled the present times as the “postgenomic era”. Proteomics approaches had revealed its utility and relevance in many studies, showing that the exclusive use of genomic approaches contributes to losses of significant information, as the proteins and not the genes coding them, are finaly responsible for the observed phenotype. In this context, we had prepared a compendium of the different aspects related to the proteomics approaches to study the interaction of plant and microbes. In this special issue, we had covered the relevance of these interactions in several ways by describing from a proteomics point of view, the relevance between roots and soil microbes, and the last advances related to virus and bacterial interactions with plants. Plant pathology has been tackled by studying the last advances in the proteomics of plant pathogenic fungi, the use of proteomics to describe bio-control agents, as well as, the development of new ways to study this relationship by using activity based proteomics probes, which had opened new frontiers beyond standard proteomics tools. The plant point of view has been included in a specific plant biotic stress contribution. Technical contributions are indispensable due to most of the used biological systems presenting poor molecular information. For this reason, the protein identification by MS/MS and the development of new gel-free approaches has been included in our special issue. We Sincerely thank the issue contributors, as they had prepared a nice compendium which will be necessary for future researchers in this field. Many of its future challenges that may likely be encountered have been described here. By summarizing, the main problem is to transfer our lab experiments to the crops field. In spite of several relevant advances, we have not yet developed any fungicide based on molecular information. We still use the chemical compounds that present several environmental problems and health risk to the farmers and consumers. The development of plants resistant to pathogen attack, new bio control agents and plant development improvement will be the areas of future proteomics research.

Mass spectrometry (MS) has become an essential technology for proteomics applications in biological sciences. Advances in this technique have been possible owing to improvements in MS instrumentation, new experimental strategies in sample preparation, and development of bioinformatics tools for data analyses. In recent years, complementary strategies to the classical two-dimensional gel electrophoresis approaches (2-DE) have been developed. These techniques are based on multidimensional peptide separation coupled to tandem MS (also referred as “second generation proteomics”), enabling protein expression analysis and high throughput protein identification studies. New methods such as Multidimensional Protein Identification Technology (MudPIT) and stable isotope labeling of protein/peptide samples (either by chemical, metabolic, or enzymatic methods), among others, are powerful tools for large-scale studies on characterization and expression of proteins in complex biological systems. Hence, these techniques can be very useful in the study of plant-pathogen interactions, aiding to detect and characterize both plant proteins concerned in defense reactions and pathogen proteins involved in pathogenicity and/or virulence. But these techniques have been implemented in these biological systems just recently. We will examine here how MS-based proteomics approaches are helping to better understand the multifaceted phenomena underlying plant-pathogen interactions.

The explosion in the amount of genomic information available has revolutionized almost every aspect of the life sciences including proteomics. In tandem with advances in genomics, considerable developments in proteomic tools and technologies have greatly facilitated the application of proteomics to tackle numerous biological questions. Many measurable characteristics of proteins, including expression levels, cellular distributions, interactions with other molecules, and post-translational modifications, influence and direct their functions in various cellular processes. Proteomic techniques, such as comparative proteomics, mass spectrometry-based identification of post-translational modifications, and protein arrays, can be employed to study these aspects of proteins in a “host plus microbes” setting and in turn shed light on their interactions. This review summarizes the proteomic techniques applicable to host-microbial relations, with a particular focus on plant-bacterial interactions. This article also provides a comprehensive overview of the applications of proteomics in plant-bacterial interactions, including some of the most recent progress in the field.

The use of affinity purification to isolate protein complexes from biological tissues, followed by mass spectrometry (AP-MS), has ballooned in recent years due to improvements in affinity purification protocols, sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, and technological advances in mass spectrometry. Plant biologists are now exploiting AP-MS to identify plausible protein-protein interactions crucial to plant defense systems. As a result, knowledge of protein interactions in plants has grown. For example, new protein partners have been found to interact with RIN4 and RPS2, two plasma membrane-bound proteins critical for defense responses in Arabidopsis thaliana. Moreover, a nuclear protein complex in A. thaliana that includes the defense signaling protein MOS4 has been affinity purified and many of the identified protein partners found to be conserved with those in a protein complex previously characterized in yeast and humans. In another example, several proteins were identified that interact with a defense signaling GTPase, Rac1, in rice (Oryza sativa). Clearly, AP-MS is an important technique that will continue to provide novel insight into protein-protein interaction networks in plants. Here we review some of these recent discoveries and summarize the different techniques of AP-MS that have been used successfully to identify some of the interacting proteins in the plant defense response to pathogen attack.

The interactions between plants and microbes have been widely studied using gene expression studies and small molecule exchange between organisms. For the most part, these studies have focused on aboveground interactions and fewer studies have examined these types of processes belowground. The purpose of this review is to summarize the current literature looking at the interactions between roots and soil microbes, with an emphasis on the exchange of proteins between the organisms. Roots can establish close contact with different microorganisms in the rhizosphere, from pathogens to beneficial bacteria such as nitrogen fixers; recent data indicate that protein exchange is an integral part of these associations. These interactions include the release by roots of defense proteins, proteins involved in bacterial chemotaxis and proteins found inside root border cells, and release of proteins from bacteria that could activate innate immunity in plants. The overall goal of this review is to convey recent proteomic information related to root-microbe exchange to identify potential areas of development to improve agriculture.

Being sessile, plants mainly depend on physiological and metabolic adaptations to obtain the phenotypic flexibility required to withstand the adverse biotic and abiotic growth conditions with which they are faced everyday. While the responses of plants to abiotic stresses are mainly focussed on maintaining cellular homeostasis, in the response to biotic stresses, the compatible or incompatible interaction between two different biotic entities, some very specific responses can be discerned. The specificity of biotic stresses, generally limited to a small group of related plant species, makes that these events cannot be studied using the established model species for plant molecular research. Therefore, an overview of the techniques and tools used for the identification of proteins in biotic stress studies will be given. The text is subdivided in two main sections, a first part focussed on an overview of the techniques that are currently used in the study of biological question using proteomics (e.g. gel- or non-gel-based techniques, posttranslational modifications or PTM) and a second part wherein some examples of biotic stress studies on plants are discussed. Because expert reviews on literature concerning the interactions of plants with the different groups of biotic stressors are part of this special issue, the emphasis is on the experimental methods and techniques used.

Trichoderma is an ascomycete fungal genus including species with a significant impact on agriculture and industrial processes. The first application of the proteome technology was carried out to extract and separate cell wall associated proteins of T. reesei. Following this work several studies performed on the proteome of different strains of T. atroviride, T. asperellum or T. harzianum interacting with pathogens and plants have provided many novel data that improve our understanding of the agricultural and biotechnological value of these fungi.

Pathogenic fungi in plants are now one of the most serious causes of crop losses in Europe. Phytopathogenic fungi are able to infect any tissue at any stage of plant growth. Most significantly, these organisms are able to maintain their infective capacity between seasons because sources of inoculum can remain present in soils and plant debris. The value of producers' crop losses and the costs of fungicide treatments are extremely relevant economic data. The complex cycle of infection is directed by a set of genes/proteins known as pathogenicity or virulence factors. Proteomics is one of the techniques that is currently attracting much interest in the study of these organisms. At present, there is a significant increase in the number of research projects in which proteomics approaches are being used to understand the complex interactions between plants and their pathogens. This trend confirms the usefulness of these techniques for studying the biology of these pathogens, their pathogenicity or virulence factors, and for exploring new proteins that could be used as therapeutic targets. However, there are still problems to be resolved in these approaches. There is also a continuous process of improvement in many crucial stages of these proteomic studies of fungal plant pathogens - in experimental designs, in protein extraction procedures, in the separation and identification of proteins, in the analysis of results of proteome studies, and more. This review will describe the different approaches being taken in proteomics studies of fungal plant pathogens. It will highlight the solutions being adopted to overcome the complications arising from the nature of the specific biological samples. Currently, there are no fungicides on the market that have been developed from molecular biology studies. Consequently the knowledge gained from past and present proteomic studies is still to be used in the design of fungicides, although the authors are optimistic in this respect.

Plant viruses exploit cellular factors, including host proteins, membranes and metabolites, for their replication in infected cells and to establish systemic infections. Besides traditional genetic, molecular, cellular and biochemical methods for studying plant-virus interactions, both global and specialized proteomics methods are emerging as useful approaches for the identification of all the host proteins that play roles in virus infections. The various proteomics approaches include measuring differential protein expression in virus-infected versus noninfected cells, analysis of viral and host protein components in the viral replicase or other virus-induced complexes, as well as proteome-wide screens to identify host protein - viral protein interactions using protein arrays or yeast two-hybrid assays. In this review, we will discuss the progress made in plant virology using various proteomics methods, and highlight the functions of some of the identified host proteins during viral infections. Since global proteomics approaches do not usually identify the molecular mechanisms of the identified host factors during viral infections, additional experiments using genetics, biochemistry, cell biology and other approaches will often prove necessary to characterize the functions of host factors. Overall, the everimproving proteomics approaches promise further understanding of plant-virus interactions that will likely result in new strategies for viral disease control in plants.

To accelerate functional annotation of proteins with a role during plant-pathogen interactions it is essential to monitor activities of proteins rather than the abundance of transcripts and proteins, since many proteins are posttranslationally regulated during antagonistic interactions. Activity-based protein profiling (ABPP) displays the active proteome using small molecule probes that react with the active site of proteins in an activity-dependent manner. ABPP is a simple and powerful functional proteomics approach that has made important contributions to studies on immune responses and plant-pathogen interactions. ABPP revealed up-regulated proteasome activities during immune responses, and displayed differential serine hydrolase activities of both host and pathogen during infection. Furthermore, ABPP in the presence of putative inhibitors demonstrated that pathogens from different kingdoms produce effectors that suppress different proteolytic activities of the host. Taken together, these examples show that ABPP is a simple and robust way to capture functional information beyond standard proteomic techniques.

The article entitled, “Protein Identification in Sub Proteome Fractions of Breast Cancer Cells by OFFGEL-IEF and iTRAQ Labeling”, by K.H. Chandramouli, Pushpa Agrawal, K.N. Thimmaiah, published in Current Proteomics, April 2009, Vol. 6, No. 1, pp. 43-50 has been unreservedly retracted by the publisher since it failed to meet our standards for accurate reporting of data.