Abstract
Guanylate kinases (GKs), which convert guanosine monophosphate into guanosine diphosphate (GDP), are important for growth and mannose outer chain elongation of cell wall N-linked glycoproteins in yeast. Here, we identified the ortholog of Saccharomyces cerevisiae GK Guk1, named MoGuk1 and a novel family of fungal GKs MoGuk2 in the rice blast fungus Magnaporthe oryzae. MoGuk1 contains 242 aa with an C-terminal GuKc domain that very similar to yeast Guk1. MoGuk2 contains 810 amino acids with a C-terminal GuKc domain and an additional N-terminal efThoc1 domain. Expression of either MoGuk1 or MoGuk2 in heterozygote yeast guk1 mutant could increase its GDP level. To investigate the biological role of MoGuk1 and MoGuk2 in M. oryzae, the gene replacement vectors were constructed. We obtained the ΔMoguk2 but not ΔMoguk1 mutant by screening over 1,000 transformants, indicating MoGuk1 might be essential for M. oryzae. The ΔMoguk2 mutant showed weak reductions in vegetative growth, conidial germination, appressorial formation, and appressorial turgor, and showed significant reductions in sporulation and pathogenicity. Moreover, the ΔMoguk2 mutant failed to produce perithecia and was sensitive to neomycin and a mixture of neomycin-tunicamycin. Exogenous GDP and ATP partially rescued the defects in conidial germination, appressorial formation, and infectious growth of the mutant. Further analysis revealed that intracellular GDP and GTP level was decreased, and GMP level was increased in the mutant, suggesting that MoGuk2 exhibits enzymatic activity. Structural analysis proved that the efThoc1, GuKc, and P-loop domains are essential for the full function of MoGuk2. Taken together, our data suggest that the guanylate kinase MoGuk2 is involved in the de novo GTP biosynthesis pathway and is important for infection-related morphogenesis in the rice blast fungus.
Highlights
Rice blast, caused by Magnaporthe oryzae, is one of the most devastating rice diseases worldwide (Yan and Talbot, 2016; Zhang et al, 2016)
We proved that MoGuk1 likely was essential for M. oryzae, and MoGuk2 is involved in the de novo guanosine triphosphat (GTP) biosynthesis pathway and is important for development and virulence in the rice blast fungus
Phylogenetic analysis for the homologs of MoGuk1 and MoGuk2 in some fungal species indicated that MoGuk1, as well as ScGuk1, formed a conserved group that contained one ortholog in each analyzed species; while MoGuk2 orthologs were only found in a few species, and formed a conserved group that different from Guk1 group, indicating MoGuk2 is a novel family of Guanylate kinase (GK) in M. oryzae (Figure 2A)
Summary
Rice blast, caused by Magnaporthe oryzae, is one of the most devastating rice diseases worldwide (Yan and Talbot, 2016; Zhang et al, 2016). To complete the infection cycle, M. oryzae evolved genetic regulatory systems that allow it to respond to available nutrients in the plant host and encounter nitrogen-starved environments at the start of the infection cycle (Snoeijers et al, 2000; Wilson et al, 2007; Fernandez et al, 2014). Many regulators (such as Nut, Npr1/2, Nir, Rbp, Snf, Tps, Nmr, and Mdt1) involved in these systems were characterized to be important for appressorial formation and penetration, as well as infection in M. oryzae (Froeliger and Carpenter, 1996; Lau and Hamer, 1996; Wilson et al, 2007, 2010, 2012; Yi et al, 2008; Franceschetti et al, 2011; Fernandez et al, 2012)
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