Some possible mechanism deployed by fungal pathogens to prevent the effects of plant PR-proteins.

Expression and accumulation of PR proteins is part of the defense machinery developed by plant to contrast plant pathogens attack. Different classes of PR-proteins accumulate locally, near the site of infection, and sistemically along the entire plant. It is supposed that plant pathogens have evolved mechanisms to avoid or prevent the harmful effects of PR-proteins. We have studied the effects of PR-proteins from grape on the fungal necrotrophic pathogens Botrytis cinerea and Sclerotium rolfsii. Mature berries and wine were chosen as useful sources of PR-proteins, especially because enriched in chitinase and thaumatin-like proteins. When the fungi were grown on berry juice, PR-proteins precipitated probably because complexes with plant oxidized polyphenols occurred, a process catalyzed by fungal laccase. When berry or wine proteins were added to the medium, B. cinerea produced appreciable proteinase activity and showed a noticeable mycelium fragmentation in comparison when grown in culture containing heat-denatured proteins. Instead, S. rolfsii grew in a normal way in the presence of native PR proteins and secreted only trace amounts of proteinase activity in the medium. The abundant glucan sheath, externally secreted by S. rolfsii, appears as the principle mechanism deployed by this fungus to defend itself from the toxic effects of grape PR proteins

Some possible mechanisms deployed by fungal pathogens to prevent the effects of plant PR-proteins

Expression and accumulation of PR proteins is part of the defense machinery developed by plant to contrast plant pathogens attack. Different classes of PR-proteins accumulate locally, near the site of infection, and sistemically along the entire plant. It is supposed that plant pathogens have evolved mechanisms to avoid or prevent the harmful effects of PR-proteins. We have studied the effects of PR-proteins from grape on the fungal necrotrophic pathogens Botrytis cinerea and Sclerotium rolfsii. Mature berries and wine were chosen as useful sources of PR-proteins, especially because enriched in chitinase and thaumatin-like proteins. When the fungi were grown on berry juice, PR-proteins precipitated probably because complexes with plant oxidized polyphenols occurred, a process catalyzed by fungal laccase. When berry or wine proteins were added to the medium, B. cinerea produced appreciable proteinase activity and showed a noticeable mycelium fragmentation in comparison when grown in culture containing heat-denatured proteins. Instead, S. rolfsii grew in a normal way in the presence of native PR proteins and secreted only trace amounts of proteinase activity in the medium. The abundant glucan sheath, externally secreted by S. rolfsii, appears as the principle mechanism deployed by this fungus to defend itself from the toxic effects of grape PR proteins

Grape berry proteins act as scavengers of grape polyphenols and protect polygalactutonase activity of Botrytis cinerea from inhibition

At maturity grape berries contain a large amount of proteins and polyphenols with potential anti-fungal activity. The protein fraction of grape extract includes mostly thaumatin-like proteins and chitinase while flavonoids predominate among polyphenols. However, Botrytis cinerea grows well in the presence of proteins and polyphenols when these molecules are supplied to the culture in a ratio similar to that measured in the grape fruit extract. In vitro it was observed that a fraction of grape proteins interacts with polyphenols forming complexes with reduced solubility and that B. cinerea laccase enhances this process. Polyphenols are well known inhibitors of fungal polygalacturonases (PGs) activity, and B. cinerea PG activity is inhibited by grape polyphenols in vitro. However, the simultaneous administration of grape proteins diminishes the inhibitory activity of grape polyphenols, and laccase addition restores completely B. cinerea PG activity. Similar results are obtained when the stylbenic phytoalexin resveratrol is used in combination with grape proteins and B. cinerea laccase. The scavenging of polyphenols by grape proteins could favour the berry infection by B. cinerea

CHARACTERIZATION OF BOTRYTIS CINEREA POLYGALACTURONASES AND LACCASES DURING GRAPE BERRIES INFECTION

Botrytis cinerea, the causal agent of grey mould disease on grapevine, when infects the grape berries encounters an environment rich in polyphenols and proteins with potential anti-fungal activity. In particular, the stilbenic phytoalexin trans-resveratrol and proteins structurally and functionally related to plant pathogenesis-related (PR) proteins, including mostly thaumatin-like proteins and chitinase. B. cinerea is thought to infect the host tissue by producing cell-wall degrading enzymes, such as polygalacturonases (PGs), and detoxification enzymes, for example laccase which is likely to be involved in trans-resveratrol detoxification. The combination of trans-resveratrol and grape polyphenols or proteins induce in vitro a strong release of B. cinerea laccase activity which, in turn, neutralizes the toxicity of grape stilbenic phytoalexins and, by oxidizing grape polyphenols, causes the insolubilization of grape proteins. This mechanism could favour berry infection by the fungus and is in accordance to the observation that grape berries infected with B. cinerea show a strong reduction of the protein content in comparison to healthy ones. Grape polyphenols have been also shown to inhibit B. cinerea PG activity in vitro. Nevertheless, the importance of B. cinerea PGs in grape berries infection cannot be ruled out since the fungus could have evolved an avoidance mechanism in order to escape the inhibitory effect of polyphenols. To better understand the role played by B. cinerea PG and laccase activities during grape berries infection, we are analyzing the enzyme activities secreted by the fungus in the infected berry and the expression of the corresponding encoding genes

The role of grape polyphenols on trans-resveratrol activity against Botrytis cinerea and of fungal laccase on the solubility of putative grape PR proteins

The necrotrophic fungus Botrytis cinerea is the causal agent of grey mould disease on grapevine. In contact with mature grape berries, the fungus encounters an environment particularly rich in polyphenols and proteins, where the stilbenic phytoalexin trans-resveratrol may accumulate. Some grape proteins are structurally and functionally related to plant pathogenesis-related proteins. To mimic conditions similar to those found in grape berries, B. cinerea was grown in vitro with proteins and polyphenols extracted from mature grapes, and with trans-resveratrol. Results showed that in the presence of highly toxic amounts of trans-resveratrol, grape polyphenols allowed total recovery of fungal growth, and proteins allowed partial recovery. These resveratrol-polyphenol or resveratrol-protein combinations also induced a strong release into the medium of laccase activity, which is likely to be involved in transresveratrol detoxification. The protein pattern changed during fungal growth; most grape proteins quickly disappeared from the culture when polyphenols and transresveratrol were present together. Similar protein patterns were obtained in vitro by incubating grape proteins with grape polyphenols and/or trans-resveratrol with a purified B. cinerea laccase. Under these conditions, most proteins became insoluble. The grape protein pattern obtained from grape berries infected by B. cinerea strongly resembled that obtained in vitro by incubating grape proteins and polyphenols with fungal laccase. It seems that B. cinerea, through laccase secretion and activity and by exploiting the berry polyphenols, easily neutralizes the toxicity of grape stilbenic phytoalexins and makes the grape pathogenesis-related proteins insoluble

Botrytis cinerea laccase abolishes resveratrol toxicity and reduces Grape PR proteins solubility

Some grape proteins are structurally and functionally related to plant pathogenesis-related proteins. When challenging mature grape berries, Botrytis cinerea encounters these PR proteins as well the stilbenic phytoalexin trans-resveratrol and other grape polyphenols. To mimic these conditions, Botrytis cinerea was grown in vitro with proteins and polyphenols extracted from mature grapes and with trans-resveratrol. Results showed that in the presence of highly toxic amounts of trans-resverattol, grape polyphenols and proteins allowed total and partial recovery of fungal growth, respectively. These resveratrol-polyphenol or resveratrol-protein combinations also induced a strong release into the medium of laccase activity, which is likely to be involved in the trans-resveratrol detoxification process. Most grape proteins quickly disappeared from the culture when polyphenols and trans-resveratrol were supplied together and similar protein patterns were obtained in vitro by incubating grape proteins with grape polyphenols and/or trans-resveratrol with a purified B. cinerea laccase. Under these conditions, most protein became insoluble. The grape protein pattern obtained from grape berries infected by B. cinerea strongly resembled that obtained in vitro by incubating the grape proteins and polyphenols with the fungal laccase. It seems that B. cinerea, in the presence of the berry polyphenols and through laccase activity, easily neutralizes the toxicity of grape stilbenic phytoalexins and makes the grape pathogenesis-related proteins insoluble. Whether changes of grape proteins solubility affect the expression of proteinase genes and the proteolytic activity of B. cinerea is currently under investigation

Necrotrophic fungal plant pathogens display different mechanisms to counteract grape chitinase and thaumatin-like protein

We characterized the ability of the necrotrophic plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Sclerotinia minor and Sclerotium rolfsii to degrade or sequester two widespread plant PR proteins: a type IV chitinase and a thaumatin-like protein (TLP). A protein (150 mg mL1) extract from grape berries, containing about 58 and 68 mg mL1 of TLP and chitinase, respectively, was added to the fungal cultures. The growth of the four fungi was not negatively affected by these proteins and, as determined by RPHPLC, both TLP and chitinase were partially removed from the medium by the three ascomycetes fungi and almost completely by the basidiomycete S. rolfsii. Different levels of protease activity were secreted by fungi but these activities were ineffective against TLP and only partially active against chitinase. The cleavage of chitinase by B. cinerea protease generated a characteristic lower molecular size band on SDS-PAGE. To verify a possible absorption of TLP and chitinase by the fungal talli, mycelia were treated with b-1,3- glucanase. TLP and, to a lower extent, chitinase were released from mycelium of the three ascomycetes fungi but not from that of S. rolfsii. The treatment with b-1,3-glucanase of a mixture containing PR proteins and a purified preparation of the S. rolfsii glucan did not release TLP or chitinase. However, the two proteins were observed when the mixture was analyzed on SDS-PAGE. This result indicates a different type of binding of PR proteins with the glucan matrix of S. rolfsii in comparison to that of the three ascomycetes. As determined by RT-qPCR, one of the two examined putative glucan synthase genes of S. rolfsii was up-regulated following the administration of PR proteins, suggesting the formation of new glucan. Overall, in comparison to protease activity, the sequestering capacity of the fungal glucan matrix seems to play a major role in the fungal defense against the plant TLP and chitinase

A Fusarium graminearum xylanase expressed during wheat infection is a necrotizing factor but is not essential for virulence

Fusarium graminearum is the fungal pathogen mainly responsible for Fusarium head blight (FHB) of cereal crops, which attacks wheat spikes, reducing crop production and quality of grain by producing trichothecene mycotoxins. Several cytohistological studies showed that spike infection is associated with the production of cell wall degrading enzymes. Wheat tissue, as in other commelinoid monocot plants, is particularly rich in xylan which can be hydrolyzed by fungal endo-1,4-beta-xylanase. The FG_03624 is one of the most expressed xylanase genes in wheat spikes 3 days after inoculation and was heterologously expressed in the yeast Pichia pastoris. The recombinant protein (22.7 kDa) possessed xylanase activity and induced cell death and hydrogen peroxide accumulation in wheat leaves infiltrated with 10 ng/mu l or in wheat lemma surface treated with 20 ng/mu l. This effect reflects that observed with other described fungal xylanases (from Trichoderma reesei, Trichoderma viride and Botrytis cinerea) with which the FG_03624 protein shares a stretch of amino acids reported as essential for elicitation of necrotic responses. Several E graminearum mutants with the FG_03624 gene disrupted were obtained, and showed about 40% reduction of xylanase activity in comparison to the wild type when grown in culture with xylan as carbon source. However, they were fully virulent when assayed by single floret inoculation on wheat cvs. Bobwhite and Nandu. This is the first report of a xylanase able to induce hypersensitive-like symptoms on a monocot plant. (C) 2013 Elsevier Masson SAS. All rights reserved