| Summary: | Cyclic diguanylate (c-di-GMP) affects bacterial physiological and biochemical functions like biofilm, motility, virulence, and bacterial secretion systems. GGDEF/EAL-domain proteins, participating in c-di-GMP synthesis and degradation, are widely present in <i>Pseudomonas</i>, with various structures and functions. <i>Pseudomonas glycinae</i> MS82 is a rhizosphere bacterium that protects mushroom against the pathogenic fungi. Although 14 genes encoding GGDEF/EAL-domain proteins have been identified in the genome of MS82, c-di-GMP regulation is poorly understood as a facilitator or repressor of physiological phenotypes. Here, <i>PafQ</i> and <i>PafR</i>, encoding the proteins with the tandem GGDEF/EAL domain, were functionally analyzed and found to regulate antifungal activity. Individual deletion mutants of <i>PafQ</i> and <i>PafR</i> were constructed in <i>P. glycinae</i> MS82 through biparental conjugation and homologous recombination. Subsequently, antifungal activity, biofilm formation, motility, and expression of the genes related to antifungal substance synthesis were examined and contrasted with those of wild-type <i>P. glycinae</i> MS82. Most phenotypes of physiological activities were significantly reduced after knocking out <i>PafQ</i> or <i>PafR</i>. In other members of the genus <i>Pseudomonas</i>, homologous genes of <i>PafQ</i> and <i>PafR</i> possess different functions in c-di-GMP regulation. In <i>P. glycinae</i>, the positive regulation of <i>PafQ</i> and <i>PafR</i> on fungistatic substance synthesis, biofilm formation, and motility is crucial in the biocontrol of mushroom diseases.
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