Boqiang Li

Crucial Role of Antioxidant Proteins and Hydrolytic Enzymes in Pathogenicity of Penicillium expansum Analysis Based on Proteomics Approach

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase.

Reactive oxygen species involved in regulating fruit senescence and fungal pathogenicity.

Senescence is a vital aspect of fruit life cycles, and directly affects fruit quality and resistance to pathogens. Reactive oxygen species (ROS), as the primary mediators of oxidative damage in plants, are involved in senescence. Mitochondria are the main ROS and free radical source. Oxidative damage to mitochondrial proteins caused by ROS is implicated in the process of senescence, and a number of senescence-related disorders in a variety of organisms. However, the specific sites of ROS generation in mitochondria remain largely unknown. Recent discoveries have ascertained that fruit senescence is greatly related to ROS and incidental oxidative damage of mitochondrial protein. Special mitochondrial proteins involved in fruit senescence have been identified as the targets of ROS. We focus in discussion on our recent advances in exploring the mechanisms of how ROS regulate fruit senescence and fungal pathogenicity.

Proteomic analysis of changes in mitochondrial protein expression during fruit senescence.

Fruit senescence has been reported to be an oxidative phenomenon, but the detailed mechanisms by which ROS regulate this process remain largely unknown. Here we show that senescence process of apple fruit was concomitant with the dynamic alterations in the mitochondrial proteome. Mitochondrial proteins involved in tricarboxylic acid cycle, electron transport chain, carbon metabolism, and stress response were found to be differentially expressed during fruit senescence. Alleviating oxidative stress by lowering the ambient oxygen concentration noticeably decreased the number of changed proteins and delayed fruit senescence, indicating the involvement of ROS in this process. To further investigate the regulatory effect of ROS on senescence process, we analyzed the mitochondrial proteome variations upon exposure to high oxygen (100%), which induces oxidative stress and accelerates fruit senescence. High oxygen treatment led to a further identification of differentially expressed proteins such as mitochondrial manganese superoxide dismutase, an antioxidant scavenging superoxide radicals produced in the mitochondria. Activity of manganese superoxide dismutase was reduced after high oxygen exposure, accompanied by an increase in oxidative protein carbonylation (damaged proteins). These data suggest that ROS may regulate fruit senescence by changing expression profiles of specific mitochondrial proteins and impairing the biological function of these proteins.

Functions of defense-related proteins and dehydrogenases in resistance response induced by salicylic acid in sweet cherry fruits at different maturity stages.

We report here a comparative analysis of sweet cherry (Prunus avium) fruits proteome induced by salicylic acid (SA) at different maturity stages. The results demonstrated that SA enhanced the resistance of sweet cherry fruits against Penicillium expansum, resulting in lower disease incidences and smaller lesion diameters, especially at earlier maturity stage. Based on proteomics analysis, 13 and 28 proteins were identified after SA treatment at earlier (A) and later (B) maturity stage, respectively. Seven antioxidant proteins and three pathogenesis related‐proteins were identified at both A and B stages, while five heat shock proteins and four dehydrogenases were only detected at B stage. SA treatment also stimulated higher transcript levels of peroxidase, but repressed that of catalase. Moreover, some proteins regulated by SA at B maturity stage were identified as enzymes involved in glycolysis and tricarboxylic acid cycle. These findings indicated that younger sweet cherry fruits showed stronger resistance against pathogen invasion after SA treatment. It further indicated that antioxidant proteins were involved in the resistance response of fruits at every maturity stage, while heat shock proteins and dehydrogenases might potentially act as factors only at later maturity stages.

Exploring Pathogenic Mechanisms of Botrytis cinerea Secretome under Different Ambient pH Based on Comparative Proteomic Analysis

Botrytis cinerea causes gray mold rot on over 200 plant species worldwide, resulting in great economic loss every year. Cooperation of proteins secreted by B. cinerea plays an important role in its successful infection to host plants. The ambient pH, as one of the most important environmental parameters, can regulate expression of secreted proteins in various fungal pathogens. In the present study, we mainly investigated the effect of ambient pH on secretome of B. cinerea strain B05.10 with a comparative proteomic method based on 2-DE. Distinct differences in secretome of B. cinerea were found between pH 4 and 6 treatments, and 47 differential spots, corresponding to 21 unique proteins, were identified using MALDI-TOF/TOF. At pH 4, more proteins related to proteolysis were induced, whereas most of up-accumulated proteins were cell wall degrading enzymes at pH 6. Analysis of gene expression using quantitative real-time PCR suggests that production of most of these proteins was regulated at the level of transcription. These findings indicate that B. cinerea can adjust protein profile of secretome responding to different ambient pH values and provide evidence to deeply understand the complicated infecting mechanisms of B. cinerea on a wide range of plant hosts.

Hydrogen peroxide acts on sensitive mitochondrial proteins to induce death of a fungal pathogen revealed by proteomic analysis.

How the host cells of plants and animals protect themselves against fungal invasion is a biologically interesting and economically important problem. Here we investigate the mechanistic process that leads to death of Penicillium expansum, a widespread phytopathogenic fungus, by identifying the cellular compounds affected by hydrogen peroxide (H2O2) that is frequently produced as a response of the host cells. We show that plasma membrane damage was not the main reason for H2O2-induced death of the fungal pathogen. Proteomic analysis of the changes of total cellular proteins in P. expansum showed that a large proportion of the differentially expressed proteins appeared to be of mitochondrial origin, implying that mitochondria may be involved in this process. We then performed mitochondrial sub-proteomic analysis to seek the H2O2-sensitive proteins in P. expansum. A set of mitochondrial proteins were identified, including respiratory chain complexes I and III, F1F0 ATP synthase, and mitochondrial phosphate carrier protein. The functions of several proteins were further investigated to determine their effects on the H2O2-induced fungal death. Through fluorescent co-localization and the use of specific inhibitor, we provide evidence that complex III of the mitochondrial respiratory chain contributes to ROS generation in fungal mitochondria under H2O2 stress. The undesirable accumulation of ROS caused oxidative damage of mitochondrial proteins and led to the collapse of mitochondrial membrane potential. Meanwhile, we demonstrate that ATP synthase is involved in the response of fungal pathogen to oxidative stress, because inhibition of ATP synthase by oligomycin decreases survival. Our data suggest that mitochondrial impairment due to functional alteration of oxidative stress-sensitive proteins is associated with fungal death caused by H2O2.

Ambient pH Stress Inhibits Spore Germination of Penicillium expansum by Impairing Protein Synthesis and Folding: A Proteomic-Based Study

Spore germination is the first step for fungal pathogens to infect host plants. The pH value, as one of the most important environmental parameters, has critical influence on spore germination. In this study, effects of ambient pH on spore germination were determined by culturing spores of Penicillium expansum in medium with pH values at 2.0, 5.0 and 8.0, and involved mechanisms were further investigated through methods of comparative proteomics. The results demonstrated that spore germination of P. expansum was obviously inhibited at pH 2.0 and 8.0. Using quadrupole time-of-flight tandem mass spectrometer, 34 proteins with significant changes in abundance were identified. Among them, 17 proteins were related to protein synthesis and folding, and most of them were down-regulated at pH 2.0 and 8.0. Accordingly, lower content of total soluble proteins and higher ratio of aggregated proteins were observed in spores at pH 2.0 and 8.0. In addition, it was found that ambient pH could affect intracellular pH and ATP level of P. expansum spores. These findings indicated that ambient pH might affect spore germination of P. expansum by changing intracellular pH and regulating protein expression. Further, impairing synthesis and folding of proteins might be one of the main reasons.

Effects of trehalose on stress tolerance and biocontrol efficacy of Cryptococcus laurentii

Aims:  To investigate the effects of internal trehalose on viability and biocontrol efficacy of antagonistic yeast Cryptococcus laurentii under stresses of low temperature (LT), controlled atmosphere (CA) and freeze drying.

Genomic Characterization Reveals Insights Into Patulin Biosynthesis and Pathogenicity in Penicillium Species

Penicillium species are fungal pathogens that infect crop plants worldwide. P. expansum differs from P. italicum and P. digitatum, all major postharvest pathogens of pome and citrus, in that the former is able to produce the mycotoxin patulin and has a broader host range. The molecular basis of host-specificity of fungal pathogens has now become the focus of recent research. The present report provides the whole genome sequence of P. expansum (33.52 Mb) and P. italicum (28.99 Mb) and identifies differences in genome structure, important pathogenic characters, and secondary metabolite (SM) gene clusters in Penicillium species. We identified a total of 55 gene clusters potentially related to secondary metabolism, including a cluster of 15 genes (named PePatA to PePatO), that may be involved in patulin biosynthesis in P. expansum. Functional studies confirmed that PePatL and PePatK play crucial roles in the biosynthesis of patulin and that patulin production is not related to virulence of P. expansum. Collectively, P. expansum contains more pathogenic genes and SM gene clusters, in particular, an intact patulin cluster, than P. italicum or P. digitatum. These findings provide important information relevant to understanding the molecular network of patulin biosynthesis and mechanisms of host-specificity in Penicillium species.

Combination of antagonistic yeasts with two food additives for control of brown rot caused by Monilinia fructicola on sweet cherry fruit

Aims:  To evaluate beneficial effect of two food additives, ammonium molybdate (NH4‐Mo) and sodium bicarbonate (NaBi), on antagonistic yeasts for control of brown rot caused by Monilinia fructicola in sweet cherry fruit under various storage conditions. The mechanisms of action by which food additives enhance the efficacy of antagonistic yeasts were also evaluated.