Virus Diseases of Peanut

Adapting their metabolism to varying carbon and nitrogen sources, saprophytic fungi produce an arsenal of extracellular enzymes, the secretome, which allows for an efficient degradation of lignocelluloses and further biopolymers. Based on fundamental advances in electrophoretic, chromatographic, and mass spectrometric techniques on the one hand and the availability of annotated fungal genomes and sophisticated bioinformatic software tools on the other hand, a detailed analysis of fungal secretomes has become feasible. While a number of reports on ascomycetous secretomes of, e.g., Aspergillus, Trichoderma, and Fusarium species are already available, studies on basidiomycetes have been mainly focused on the two model organisms Phanerochaete chrysosporium and Coprinopsis cinerea so far. Though an impressive number and diversity of fungal biocatalysts has been revealed by secretome analyses, the identity and function of many extracellular proteins still remains to be elucidated. A comprehensive understanding of the qualitative and quantitative composition of fungal secretomes, together with their synergistic actions and kinetic expression profiles, will allow for the development of optimized enzyme cocktails for white biotechnology.

Fungal secretomes—nature’s toolbox for white biotechnology

https://www.itqb.unl.pt/~imartins/Kim_etal_2007.pdf

Proteomics of filamentous fungi

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2-D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC-MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.

The Botrytis cinerea early secretome.

Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.

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Proteomics of Plant Pathogenic Fungi

In order to gain a more thorough understanding of the phytopathogenic fungus, Sclerotinia sclerotiorum, we initiated a proteome-level study of the fungal mycelia and secretome. To our knowledge, this is the first comprehensive proteome-level study of this fungus. Extracted mycelial proteins and secreted proteins collected from liquid culture were separated using 2-DE and annotated following ESI-q-TOF MS/MS. Fifty-two secreted proteins were reproducibly present in three biological replicates and 18 of them were identified by MS/MS while over 200 mycelial proteins were reproducibly present in three independent extractions and approximately half of them were identified. Many of the annotated secreted proteins were cell wall degrading enzymes that had been previously identified as pathogenicity or virulence factors of S. sclerotiorum; however, the contribution to the virulence of S. sclerotiorum of one of the identified proteins, alpha-L-arabinofuranosidase, is yet to be analyzed. Furthermore, previous comprehensive EST studies did not detect the presence of the alpha-L-arabinofuranosidase transcript, which demonstrates the merit of performing proteome-level research. All of the secreted and mycelial proteins identified were functionally classified, and the known and proposed roles in disease initiation or progression for many of them are discussed.

The proteome of the phytopathogenic fungus Sclerotinia sclerotiorum.

With the completion of genome projects for Arabidopsis thaliana, Oryza sativa and several other plant species, an increasing number of whole genome sequences are now available for plants. In this post-genomic era, a more thorough understanding of gene expression and function can be achieved through the characterization of the products of expression, the proteins, which are essential biological determinants of plant phenotypes. Proteomics offers a continually evolving set of novel techniques to study all facets of protein structure and function. The application of proteomics in plant pathology is becoming more commonplace with techniques such as two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) being used to characterize cellular and extracellular virulence and pathogenicity factors produced by pathogens as well as to identify changes in protein levels in plant hosts upon infection by pathogenic organisms and symbiotic counterparts. This review article summarizes the current status of geland non gel-based proteomic techniques and describes the significant discoveries that have resulted from the various proteome-level investigations into phytopathogenic microorganisms and plant host-microbe interactions.

Application of Proteomics to Investigate Plant-Microbe Interactions

Sclerotinia stem rot is an economically important disease of canola (Brassica napus) and is caused by the fungal pathogen Sclerotinia sclerotiorum. This study evaluated the differential gene expression patterns of S. sclerotiorum during disease development on two canola lines differing in susceptibility to this pathogen. Sequencing of the mRNA libraries derived from inoculated petioles and mycelium grown on liquid medium generated approximately 164 million Illumina reads, including 95 million 75-bp-single reads, and 69 million 50-bp-paired end reads. Overall, 36% of the quality filter-passed reads were mapped to the S. sclerotiorum reference genome. On the susceptible line, 1301 and 1214 S. sclerotiorum genes were differentially expressed at early (8-16 hours post inoculation (hpi)) and late (24-48 hpi) infection stages, respectively, while on the resistant line, 1311 and 1335 genes were differentially expressed at these stages, respectively. Gene ontology (GO) categories associated with cell wall degradation, detoxification of host metabolites, peroxisome related activities like fatty acid s-oxidation, glyoxylate cycle, oxidoreductase activity were significantly enriched in the up-regulated gene sets on both susceptible and resistant lines. Quantitative RT-PCR of six selected DEGs further validated the RNA-seq differential gene expression analysis. The regulation of effector genes involved in host defense suppression or evasion during the early infection stage, and the expression of effectors involved in host cell death in the late stage of infection provide supporting evidence for a two-phase infection model involving a brief biotrophic phase during early stages of infection. The findings from this study emphasize the role of peroxisome related pathways along with cell wall degradation and detoxification of host metabolites as the key mechanisms underlying pathogenesis of S. sclerotiorum on B. napus.

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Transcriptome analysis of the plant pathogen Sclerotinia sclerotiorum interaction with resistant and susceptible canola (Brassica napus) lines

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it’s in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus).

Although Sclerotinia sclerotiorum (Lib.) de Bary has been studied extensively, there are still aspects of this important phytopathogen's ability to cause disease in susceptible plants that remain unclear. A recent comprehensive proteome-level investigation of this fungus identified a number of proteins whose functions in disease initiation and progression have not been clearly established. Included among these proteins was an arabinofuranosidase/beta-xylosidase precursor whose role as a potential virulence factor had not been investigated previously. This article describes the generation of gene-disrupted mutant S. sclerotiorum unable to produce this arabinofuranosidase/beta-xylosidase precursor as well as the comparison of the virulence of this mutant with that of wild-type mycelia on susceptible canola leaves and stems. At all time points tested, the degree of necrosis was observed to be significantly greater on the plant tissue inoculated with wild-type mycelia. To our knowledge, this is the first report that clearly demonstrates that this arabinofuranosidase/beta-xylosidase precursor is a virulence factor for S. sclerotiorum.

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-22-7-0783

William Yajima

Papers

The proteome of the phytopathogenic fungus Sclerotinia sclerotiorum.

Application of Proteomics to Investigate Plant-Microbe Interactions

Transcriptome analysis of the plant pathogen Sclerotinia sclerotiorum interaction with resistant and susceptible canola (Brassica napus) lines

A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus).

Gene Disruption of an Arabinofuranosidase/β-Xylosidase Precursor Decreases Sclerotinia sclerotiorum Virulence on Canola Tissue