A C2H2 Zinc Finger Protein PlCZF1 Is Necessary for Oospore Development and Virulence in Peronophythora litchii.

C2H2 zinc finger is one of the most common motifs found in the transcription factors (TFs) in eukaryotes organisms, which have a broad range of functions, such as regulation of growth and development, stress tolerance and pathogenicity. Here, PlCZF1 was identified to encode a C2H2 zinc finger in the litchi downy blight pathogen Peronophythora litchii. PlCZF1 is conserved in P. litchii and Phytophthora species. In P. litchii, PlCZF1 is highly expressed in sexual developmental and early infection stages. We generated Δplczf1 mutants using the CRISPR/Cas9 method. Compared with the wild type, the Δplczf1 mutants showed no significant difference in vegetative growth and asexual reproduction, but were defective in oospore development and virulence. Further experiments revealed that the transcription of PlM90, PlLLP and three laccase encoding genes were down-regulated in the Δplczf1 mutant. Our results demonstrated that PlCZF1 is a vital regulator for sexual development and pathogenesis in P. litchii.

The Mitogen-Activated Protein Kinase PlMAPK2 Is Involved in Zoosporogenesis and Pathogenicity of Peronophythoralitchii.

As an evolutionarily conserved pathway, mitogen-activated protein kinase (MAPK) cascades function as the key signal transducers that convey information by protein phosphorylation. Here we identified PlMAPK2 as one of 14 predicted MAPKs encoding genes in the plant pathogenic oomycete Peronophythora litchii. PlMAPK2 is conserved in P.litchii and Phytophthora species. We found that PlMAPK2 was up-regulated in sporangium, zoospore, cyst, cyst germination and early stage of infection. We generated PlMAPK2 knockout mutants using the CRISPR/Cas9 method. Compared with wild-type strain, the PlMAPK2 mutants showed no significant difference in vegetative growth, oospore production and sensitivity to various abiotic stresses. However, the sporangium release was severely impaired. We further found that the cleavage of the cytoplasm into uninucleate zoospores was disrupted in the PlMAPK2 mutants, and this developmental phenotype was accompanied by reduction in the transcription levels of PlMAD1 and PlMYB1 genes. Meanwhile, the PlMAPK2 mutants exhibited lower laccase activity and reduced virulence to lychee leaves. Overall, this study identified a MAPK that is critical for zoosporogenesis by regulating the sporangial cleavage and pathogenicity of P.litchii, likely by regulating laccase activity.