Mechanistic details into NADPH oxidase mediated resistance against <em>Heterodera schachtii </em>in<em> Arabidopsis thaliana</em>

Abstract

The oxidative burst, production of Reactive Oxygen Species (ROS) in response to pathogen attack, is an important component of plant defence mechanisms. It is now generally recognised that the major source of ROS during an “oxidative burst” in plants is plasma membrane-bound NADPH oxidase. Plant NADPH oxidases have been named as respiratory burst oxidase homolog (Rboh) and possess six transmembrane-spanning domains corresponding to the domains that have been identified in the gp91^phox subunit in mammals. Genetic analysis of plants disrupted in Rboh functions suggested that they are required for the production of a full oxidative burst in response to a variety of pathogens. Yet, this lack of ROS production by NADPH oxidase has variable effects on plant responses to pathogens in terms of cell death and resistance. On one hand, it is positively correlated with plant resistance by strengthening of cell walls via cross linking, lipid peroxidation, membrane damage and activation of defence genes. On the other hand, it is an important susceptibility factor for successful infection of plants by various pathogens. Nevertheless, the mechanistic details on the pathosystem-specific role of Rboh-mediated ROS are not yet known. <br /><em>Heterodera schachtii </em>is a cyst nematode that establishes a long-term biotrophic relationship with the roots of sugar beets and brassicaceaous plants, including <em>Arabidopsis thaliana</em>. Infective stage juveniles of cyst nematodes (J2s) invade the roots and move towards the vascular cylinder where they establish a syncytial nurse cell system. The syncytium is the only source of nutrients for nematodes throughout their life-span of several weeks. This thesis focuses on the characterization of the role of Rboh-mediated ROS in plant-nematode interactions using the <em>Arabidopsis thaliana – H. schachtii</em> model system. <br /> In Arabidopsis, Rboh is encoded by ten genes (RbohA-RbohJ). We used loss-of-function mutants for Rboh genes and found that the number of female nematodes, the size of female nematodes, and the size of female-associated syncytium decreased greatly in <em>rbohD </em>and<em> rbohD/F</em>, but not in <em>rbohA, rbohB, rbohC, rbohE, rbohF, rbohG, rbohH </em>and<em> rbohJ</em> plants compared with Col-0 plants. A detailed microscopic, molecular and biochemical analysis showed that Rboh-dependent ROS are not required for root invasion; however, the syncytial establishment and development is impaired in the absence of ROS. To understand the mechanism underlying the Rboh-mediated reduced susceptibility to nematodes, we performed a genome-wide comparative transcriptome analysis between Col-0 and <em>rbohD/F</em> during early stages of infection. The gene that was most strongly downregulated encodes the vacuolar auxin transporter, Walls Are Thin 1 (WAT1). Genetic disruption of WAT1 led to similar changes in nematode susceptibility as in <em>rbohD/F</em>, thus suggesting that Rboh-mediated reduction in susceptibility to nematode is dependent on WAT1. Interestingly, both rbohD/F and wat1 are impaired in expression of key metabolic genes for indole metabolism including auxin biosynthesis upon nematode infection. In summary, our work provides for the first time a link between Rboh-mediated phenotypes and auxin metabolism. These data are the basis for a mechanistic understanding on the role of ROS as signals to promote nematode and other pathogen infections.