Proteomic and phosphoproteomic approaches to understand plant-pathogen interactions

The regulation mechanisms of any plant-pathogen interaction are complex and dynamic. A proteomic approach is valuable in understanding regulatory networks because it deals with identifying new proteins in relation to their function and ultimately aims to unravel how their expression and modification is controlled. One of the major control mechanisms for protein activity in plant-pathogen interactions is protein phosphorylation. However, studying protein phosphorylation cascades in plants presents two major technical challenges. The first is that many of the signaling components are present at very low copy numbers, which makes them difficult to detect. The second is that they are difficult to identify because there are currently only three plants with a complete genome sequence, i.e. Arabidopsis thaliana, Populus (poplar) and Oryza sativa (rice). In this article, we review proteomic approaches to study plant-pathogen interactions in both model and non-model plants and demonstrate that current proteomic and phosphoproteomic technologies have the potential to identify new components of regulatory pathways and elucidate their functions within a cellular context

Identification and characterization of a serine protease from wheat leaves

A putative serine protease with a potential role in the plant biotic and abiotic stress response was purified from wheat leaf apoplastic fluid and partially characterized. Following two-dimensional electrophoresis a protein of Mr = 75 k and a pI of 4.2 to 4.5 was observed. This protein displayed in-gel protease activity and was specifically inhibited by phenylmethanesulfonyl fluoride and partially inhibited by Ca2+ and Zn2+, but not by E-64 or leupeptin. An internal tryptic fragment of 13 amino acids was identified by MALDI QqTOF MS/MS, and this peptide showed a high level of homology (85–100 % identity) to a highly conserved region of known plant subtilisin-like proteases. We demonstrated that the protease activity increased until a late stage of wheat leaf development and increased in response to heat shock. In both cases Rubisco large subunit was degraded with time. Protease activity was also increased during biotic stress. Leaves challenged with leaf rust (Puccinia triticina), showed an approximately three fold increase in protease activity during an incompatible interaction, compared to activity in mock-inoculated leaves and to leaves in a compatible leaf rust interaction. These results suggest that the expression of this serine protease could be involved in the defense response against both abiotic and biotic stresses

Beyond R genes: Dissecting disease-resistance pathways using genomics and proteomics

Breeding for disease resistance in crops has mainly been accomplished by incorporating single resistance (R) genes. There are advantages to quantitative resistance in terms of durability but breeding for this type of resistance is difficult. New technologies in genomics and proteomics are providing insights into disease-resistance pathways. Structural genomics can identify genomic regions that carry genes controlling these pathways and provide a means for identifying and cloning the genes involved. High-throughput molecular breeding can be used to rapidly assess a large number of lines and select for multiple-resistance quantitative trait loci. This makes breeding for complex resistance types feasible. Functional genomics can identify genes involved in the resistance pathway. By merging structural and functional genomics it will be possible to correlate complex patterns of gene expression with genomic regions and identify key elements that control entire pathways. To fully understand the pathways it is necessary to look at post-translational modification of proteins, as this is a fu

Knockout of AtMKK1 enhances salt tolerance and modifies metabolic activities in Arabidopsis

Mitogen-activated protein kinase (MAPK) pathways represent a crucial regulatory mechanism in plant development. The ability to activate and inactivate MAPK pathways rapidly in response to changing conditions helps plants to adapt to a changing environment. AtMKK1 is a stress response kinase that is capable of activating the MAPK proteins AtMPK3, AtMPK4 and AtMPK6. To elucidate its mode of action further, several tests were undertaken to examine the response of AtMKK1 to salt stress using a knockout (KO) mutant of AtMKK1. We found that AtMKK1 mutant plants tolerated elevated levels of salt during both germination and adulthood. Proteomic analysis indicated that the level of the α subunit of mitochrondrial H+-ATPase, mitochrondial NADH dehydrogenase and mitochrondrial formate dehydrogenase was enhanced in AtMKK1 knockout mutants upon high salinity stress. The level of formate dehydrogenase was further confirmed by immunoblotting and enzyme assay. The possible involvement of these enzymes in salt tolerance is discussed

Proteomic profiling reveals insights into Triticeae stigma development and function

To our knowledge, this study represents the first high-throughput characterization of a stigma proteome in the Triticeae. A total of 2184 triticale mature stigma proteins were identified using three different gel-based approaches combined with mass spectrometry. The great majority of these proteins are described in a Triticeae stigma for the first time. These results revealed many proteins likely to play important roles in stigma development and pollen-stigma interactions, as well as protection against biotic and abiotic stresses. Quantitative comparison of the triticale stigma transcriptome and proteome showed poor correlation, highlighting the importance of having both types of analysis. This work makes a significant contribution towards the elucidation of the Triticeae stigma proteome and provides novel insights into its role in stigma development and function

TAB2, a nucleoside diphosphate protein kinase, is a component of the tMEK2 disease resistance pathway in tomato

Signal transduction is used by plants to coordinate their development and to sense and respond to fluctuations in their surroundings. With previous proteomics approaches, we specifically studied activation events downstream of tMEK2, a mitogen-activated protein kinase kinase (MAPKK), in tomato. LC-MS/MS revealed a group of phosphoproteins in tMEK2MUT-transgenic tomato plants, where tMEK2 was constitutively activated. Of particular interest is TAB2

MALDI-Qq-TOF-MS and transient gene expression analysis indicated co-enhancement of β-1,3-glucanase and endochitinase by tMEK2 and the involvement of divergent pathways

A mitogen-activated protein kinase (MAPK) pathway has been demonstrated as a key pathway in plant defense against pathogen attacks. With proteomics approaches, we specifically studied activation events downstream of a MAPK kinase, tMEK2, in tomato. Overexpression of a constitutively activated tomato MAPK kinase gene (tMEK2MUT) enhanced resistance of transgenic tomato lines to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogenesis-related genes, PR1b1, β-1,3-glucanase, and endochitinase were up-regulated by tMEK2MUT. Two-dimensional electrophoresis and matrix-assisted l

Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum

NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys325) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys325 to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta