Investigating the role of NADPH oxidases during infection related development of the rice blast fungus Magnaporthe oryzae

Abstract

Rice blast disease is a major threat to global food security, destroying 30% of annual rice crops globally, and with disease remaining difficult to control. The causal agent of rice blast, Magnaporthe oryzae infects plants with a specialised single-celled infection structure called an appressorium, which accumulates glycerol to develop enormous intracellular turgor, using osmotic pressure to drive a rigid penetration peg through the rice leaf cuticle. NADPH oxidases (Nox) are flavoenzymes that function by transferring electrons across biological membranes to catalyse the reduction of molecular oxygen to superoxide. In filamentous fungi, Nox are necessary for cellular differentiation associated with sexual reproduction and host tissue invasion, such as processes involving the transition of cell growth from non-polarized growth to polarized growth. Previously, in M. oryzae it has been shown that Nox are essential for septin-mediated re-orientation of the F-actin cytoskeleton to facilitate penetration peg formation and plant cell invasion. The mechanism of assembly and activation of the Nox complex at the plasma membrane in M. oryzae is still unknown. Here I show the assembly of the Nox complex by determining the physical interactions of putative Nox associated proteins using yeast two-hybrid analysis and models of these interactions are presented. Yeast two-hybrid analysis revealed evidence of interactions between putative Nox components and the cytoskeleton element Chm1, which suggests the Nox complex may have a role in cytoskeleton remodeling during penetration peg formation. Analysis also revealed a potential scaffold protein for the recruitment of soluble proteins to the membrane bound Nox complex, Bem1, as the protein interacted with all putative Nox components investigated. In addition I generated a Δpls1 mutant using a one-step gene replacement procedure. I confirmed that the mutant is unable to cause rice blast disease.