Cpkk1, MAPKK of Cryphonectria parasitica, Is Necessary for Virulence on Chestnut

Abstract

The role of Cpkk1, a mitogen-activated protein kinase from Cryphonectria parasitica, was investigated by generating a number of mutant strains that overexpress, under the control of the cryparin promoter, both the wild-type protein and its allele with an extensive deletion in the catalytic domain. Furthermore, a hairpin construct was built and expressed to cause specific silencing of Cpkk1 mRNA transcripts. Specific mRNA silencing or overexpression was confirmed on both Northern and Western blot analysis. Selected C. parasitica strains with Cpkk1 either silenced or overexpressed were evaluated for their biological characteristics, including virulence on European chestnut, growth on different substrates, conidial sporulation, and resistance to cell-wall-degrading enzymes. Silencing of Cpkk1 and the overexpression of a defective Cpkk1 correlated with a marked reduction in virulence on 3-year-old chestnut trees, with no statistically significant effect on fungal growth in the various conditions tested.

The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high‐osmolarity glycerol signaling pathways

“…The functions of Slt2 MAPK cascade in virulence have also been characterized in several plant pathogens. Silencing of Cpkk1 (a MAPKK) in Cryphonectria parasitica correlated with a marked reduction in virulence on 3-year-old chestnut trees (Rostagno et al, 2010). In C. lagenarium, Maf1 is required for the early differentiation phase of appressorium formation because mutants failed to form appressoria (Kojima et al, 2002).…”

Functional characterization of the three mitogen‐activated protein kinase kinases (MAP2Ks) present in the Cryphonectria parasitica genome reveals the necessity of Cpkk1 and Cpkk2, but not Cpkk3, for pathogenesis on chestnut (Castanea spp.)

“…In contrast, no elements of the cell integrity pathway have been investigated so far in C. parasitica , except Cpkk1, whose function was recently revealed in our laboratory using silencing techniques (Rostagno et al ., ). Experiments demonstrated that Cpkk1 plays an important role in conidiation, growth on minimal medium and virulence.…”

“…Virulence assays were performed on stems of European chestnut tree ( Castanea sativa ) with four replicates per fungal strain as described previously (Rostagno et al ., ). Inoculated stems were kept at 24 °C under normal light covered with plastic bags to maintain moisture.…”

“…The hph cassette was obtained from the plasmid pCB1004 (Carroll et al ., ) after digestion with Hpa I, and introduced into a fragment of the coding sequence of each MAP2K amplified by PCR. Primers were designed on the basis of known MAP2K sequences (Rostagno et al ., ; Turina et al ., ) (http://onlinelibrary.wiley.com/doi/10.1111/mpp.12111/suppinfo, see Supporting Information). Briefly, MAP2K amplified fragments were cloned into the TOPO TA Vector (Invitrogen Life Technology, Carlsbad, CA, USA), digested with a specific single‐site‐cutting enzyme ( Nru I for cpkk1 , Sna BI for cpkk2 and Bsa BI for cpkk3 ) and fused with the 1.4‐kb hph cassette.…”

Comparative Methylome Analysis Reveals Perturbation of Host Epigenome in Chestnut Blight Fungus by a Hypovirus

“…Relevant to our findings, previous studies have suggested that signal transduction components may be perturbed by hypovirus infection to induce hypovirulence (Turina and Rostagno, 2007 ). Several of these genes constitute the G-protein signaling cascades while others function in mitogen-activated protein kinase (MAPK) pathways (Rostagno et al, 2010 ). MAPKs of Ser/Thr kinases have been shown to be required for fungal growth, development, and pathogenicity (Park et al, 2012 ).…”