Multiple Mutations in SDHB and SDHC2 Subunits Confer Resistance to the Succinate Dehydrogenase Inhibitor Cyclobutrifluram in Fusarium fujikuroi.

Fusarium fujikuroi is one of the dominant phytopathogenic fungi causing rice bakanae disease worldwide. Cyclobutrifluram is a novel succinate dehydrogenase inhibitor (SDHI), which shows strong inhibitory activity against F. fujikuroi. The baseline sensitivity of 112 F. fujikuroi to cyclobutrifluram was determinated with a mean EC50 value of 0.025 µg/mL. A total of 17 resistant mutants were obtained by fungicide adaptation and displayed equal or slightly weaker fitness than parental isolates, which suggests that the resistance risk of F. fujikuroi to cyclobutrifluram is medium. A positive cross-resistance was detected between cyclobutrifluram and fluopyram. The amino acid substitutions H248L/Y of FfSdhB and G80R or A83V of FfSdhC2 conferred cyclobutrifluram resistance in F. fujikuroi, which was validated by molecular docking and protoplast transformation. The results indicate that the affinity between cyclobutrifluram and FfSdhs obviously decreased after point mutations, causing the resistance of F. fujikuroi.

PcCesA1 is involved in the polar growth, cellulose synthesis, and glycosidic linkage crosslinking in the cell wall of Phytophthora capsici.

Phytophthora capsici is a destructive plant pathogen that infects a wide range of hosts worldwide. The P. capsici cell wall, rich in cellulose, is vital for hyphal growth and host interactions. However, the enzymes involved in its synthesis remain largely unelucidated. In the current study, we functionally characterized the cellulose synthase gene PcCesA1, which is highly conserved in Phytophthora. By using CRISPR/Cas9-mediated gene replacement and in situ complementation system, it was found PcCesA1 is essential for the mycelial growth, cystospore germination, and pathogenicity of P. capsici. The normal deposition of newly synthesized cell wall components and the polar growth point formation were disrupted in PcCesA1 knockout mutants, suggesting that PcCesA1 plays an important role in the polar growth of P. capsici. Compared with the wild-type strains, PcCesA1 knockout mutants displayed a thicker inner layer cell wall and were more sensitive to carboxylic acid amide fungicides (CAAs). The contents of the cell wall polysaccharides 1,4-Glc, 1,4,6-Glc, and 1,3,4-Glc were reduced in PcCesA1 knockout mutants, suggesting that PcCesA1 affected cellulose content and glycosidic linkage crosslinking in the cell wall. Our findings demonstrate that PcCesA1 is required for cell wall biogenesis. Therefore, PcCesA1 may be a potential target for Phytophthora disease control.

A Patched-Like Protein PsPTL Is Not Essential for the Growth and Response to Various Stresses in Phytophthora sojae.

Patched (Ptc) and Patched-related (Ptr) proteins containing sterol-sensing domains (SSD) and Patched domains are highly conserved in eukaryotes for lipid transport and metabolism. Four proteins containing predicted SSD and Patched domains were simultaneously found by searching the Phytophthora sojae genome database, and one of them was identified as a Patched-like (PTL) protein. Here, we investigated the biological function of PsPTL. The expression level of PsPTL was higher during mycelial and sporulation stages, compared to zoospore (ZO), cyst, and germinated-cyst stages, without significant change during infection. However, deletion of PsPTL using CRISPR/Cas9 had no significant effect on the growth, development, or virulence of P. sojae. Further investigations showed that PsPTL is not essential for P. sojae to cope with external stresses such as temperature, pH, oxidative and osmotic pressure. In addition, this gene did not appear to play an essential role in P. sojae's response to exogenous sterols. The transcript levels of the other three proteins containing predicted SSD and Patched domains were also not significantly upregulated in PsPTL deletion transformants. Our studies demonstrated that PsPTL is not an essential protein for P. sojae under the tested conditions, and more in-depth research is required for revealing the potential functions of PsPTL under special conditions or in other signaling pathways.