Correction: Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development, Virulence and Multi-Stress Tolerance in <i>Botrytis cinerea</i>

Correction: Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development, Virulence and Multi-Stress Tolerance in <i>Botrytis cinerea</i

Phosphorylation levels of BcSak1 in 38B1, ΔBcPtpA-10, and ΔBcPtpB-4.

<p>Mycelia of each strain were treated with 0.5 M NaCl or 24 mM H₂O₂ for 2 hours after being grown in potato dextrose broth for 2 days. The cultures without any treatment were used as the control (NT). BcSak1 and phosphorylated BcSak1 proteins were detected using the yeast anti-Hog1p (C-terminal anti-Hog1) and phosphorylated p38 (Thr180/Tyr182) antibodies, respectively.</p

Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development, Virulence and Multi-Stress Tolerance in <i>Botrytis cinerea</i>

<div><p>Tyrosine phosphorylation and dephosphorylation have emerged as fundamentally important mechanisms of signal transduction and regulation in eukaryotic cells, governing many processes, but little has been known about their functions in filamentous fungi. In this study, we deleted two putative protein tyrosine phosphatase (PTP) genes (<i>BcPTPA</i> and <i>BcPTPB</i>) in <i>Botrytis cinerea</i>, encoding the orthologs of <i>Saccharomyces cerevisiae</i> Ptp2 and Ptp3, respectively. Although BcPtpA and BcPtpB have opposite functions in conidiation, they are essential for sclerotial formation in <i>B. cinerea</i>. <i>BcPTPA</i> and <i>BcPTPB</i> deletion mutants ΔBcPtpA-10 and ΔBcPtpB-4 showed significantly increased sensitivity to osmotic and oxidative stresses, and to cell wall damaging agents. Inoculation tests showed that both mutants exhibited dramatically decreased virulence on tomato leaves, apples and grapes. In <i>S. cerevisiae</i>, it has been shown that Ptp2 and Ptp3 negatively regulate the high-osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway. Although both BcPtpA and BcPtpB were able to inactive Hog1 and Mpk1 in <i>S. cerevisiae</i>, in contrast to <i>S. cerevisiae</i>, they positively regulate phosphorylation of BcSak1 (the homologue of Hog1) and BcBmp3 (the homologue of Mpk1) in <i>B. cinerea</i> under stress conditions. These results demonstrated that functions of PTPs in <i>B. cinerea</i> are different from those in <i>S. cerevisiae</i>, and BcPtpA and BcPtpB play important roles in regulation of vegetative development, virulence and in adaptation to oxidative, osmotic and cell-wall damage stresses in <i>B. cinerea</i>.</p> </div

Comparisons in conidiation among 38B1, ΔBcPtpA-10, ΔBcPtpB-4, BcPtpA-5 and ΔBcPtpB-C1.

<p>(<b>A</b>) Colony morphology of the wild-type strain 38B1 and the mutants on sterilized cucumber fragments. The photos were taken after 10 days of incubation on sterilized cucumber fragments. (<b>B</b>) Quantification of conidia for each strain. The conidia of 38B1, ΔBcPtpA-10, ΔBcPtpB-4, BcPtpA-5 and ΔBcPtpB-C1 were washed off from each PDA plate after 10 days of incubation, and were counted under a microscope. Bars denote standard errors from three replications. Values on the bars followed by the same letter are not significantly different at <i>P = </i>0.05.</p

Comparison in phosphorylation of Hog1 and Mpk1 in the <i>S. cerevisiae</i> strains BY4741+ pYES2, BY4741ΔPTP2+ pYES2, BY4741ΔPTP2+pYES2-BcPTPA and BY4741ΔPTP2+pYES2-BcPTPB.

<p>Hog1 and phosphorylated Hog1 proteins were detected using the yeast anti-Hog1p (C-terminal anti-Hog1) and phosphorylated p38 (Thr180/Tyr182) antibodies, respectively.</p

Sensitivity of 38B1, ΔBcPtpA-10, ΔBcPtpB-4, BcPtpA-5 and ΔBcPtpB-C1 to the cell-wall-degrading enzymes.

<p>(<b>A</b>) Fungal mycelia of each strain were cultivated in YEPD medium for 28 h, washed and incubated for 2 h in osmotically stabilized solution (0.6 M KCl) containing 0.25% Glucanex before microscopic examination. (<b>B</b>) Protoplasts were counted microscopically after filtration from the remaining mycelium. Bars denote standard errors from three replications. Values on the bars followed by the same letter are not significantly different at <i>P = </i>0.05.</p

Phosphorylation levels of BcBmp3 in 38B1, ΔBcPtpA-10, ΔBcPtpB-4.

<p>Mycelia of each strain were treated with 0.3 mg/ml Congo red for 2 hours after being grown in potato dextrose broth for 2 days. The cultures without any treatment were used as the control (NT). BcBmp3 and phosphorylated BcBmp3 proteins were detected using the yeast anti-Mpk1 (yN-19) and phospho-p44/42 MAP kinase antibody (Cell Signaling) antibodies, respectively.</p

Complementation of <i>S. cerevisiae PTP2</i> and <i>PTP3</i> mutants with <i>BcPTPA</i> and <i>BcPTPB</i>.

<p>The <i>S. cerevisiae PTP2</i> and <i>PTP3</i> mutants were transformed with <i>BcPTPA</i> and <i>BcPTPB</i> cDNA to generate the strain BY4741ΔPTP2+pYES2-BcPTPA, BY4741ΔPTP2+pYES2-BcPTPB, BY4741ΔPTP3+pYES2-BcPTPA and BY4741ΔPTP3+pYES2-BcPTPB. The wild-type strain BY4741, BY4741ΔPTP2 and BY4741ΔPTP3 transformed with empty pYES2 vector were used as controls. Serial dilutions of cell suspension of each strain were spotted on YPRG plates under different stresses. After yeast cells were incubated at 30°C for four days, growth of each strain on each plate was examined.</p

Sensitivity of 38B1, ΔBcPtpA-10, ΔBcPtpB-4, BcPtpA-5 and ΔBcPtpB-C1 to the cell wall-damaging agents Congo red (CR) and caffeine (CF).

<p>Bars denote standard errors from three replications.</p

Involvement of <i>BcPTPA</i> and <i>BcPTPB</i> in the regulation of hypal melanization.

<p>(<b>A</b>) Comparisons of mycelial pigmentation among the wild-type strain 38B1, ΔBcPtpA-10, ΔBcPtpB-4, BcPtpA-5 and ΔBcPtpB-C1 after 9 days of incubation on PDA plates amended with or without 50 µg/ml tricyclazole. (<b>B</b>) Relative expression level of <i>THR1</i>, 1,3,8-trihydroxynaphthalene reductase gene, which is involved in melanin biosynthesis. Bars denote standard errors from three replications. Values on the bars followed by the same letter are not significantly different at <i>P = </i>0.05.</p