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Regulation of the production of reactive oxygen species by the plant NADPH oxidase and its role in pathogen response and in response to other environmental or developmental factors

Final Report Summary - OXYREG (Regulation of the production of reactive oxygen species by the plant NADPH oxidase and its role in pathogen response and in response to other environmental factors)

Project objectives

The production of reactive oxygen species (ROS) is one of the earliest responses observed after pathogen infection in plants. ROS are not direct killers but are signals that mediate the activation of the defences. In addition, plants use ROS-derived signals in a variety of developmental contexts and in abiotic stress. It is therefore important to understand the signalling controls that allow plant cells to interpret ROS-dependent signals.

Our goal here is to decipher, using functional genomics tools, the functions of the plant NADPH oxidase gene family. Different members of the respiratory burst oxidase homologues (RBOH) family, components of the plant NADPH oxidase, control the production of ROS during defence and other responses. We are performing this research using Arabidopsis, a model organism for studies in plants, where many genomic tools are available.

We propose the following two specific aims:

1. to Investigate the regulation of RBOH-dependent NADPH oxidase activity and its function in control of ROS production, pathogen response and other responses;
2. to examine the regulation of the plant NADPH oxidase by heterotrimeric G-proteins, especially in the context of defence response and cell death.

Work performed

We and others have shown that members of the RBOH-NADPH oxidase family are responsible for the production of ROS in many contexts. Interestingly, whereas AtrbohC specifically regulates root hair formation, AtrbohD and AtrbohF seem to be pleiotropic, functioning in defence response, cell death control and several stress responses. In addition, AtrbohD and AtrbohF display functional overlap, since the double mutant always displays stronger phenotypes than either of the individual mutants. But they also clearly perform independent functions. Thus, ROS produced by different Atrboh may have a qualitative (location, time, etc.) difference that may serve diverse signalling functions in different situations. To unravel the specificity of the function of ROS produced by the Atrboh, we generated different constructs with AtrbohD and AtrbohF alleles that were driven by a strong promoter or by their own promoters. These alleles carry modifications in the different regulatory domains via a fluorescent tag which follows its accumulation and cell localisation in planta. Its functionality in planta is checked by a complementation of mutant lines. The assays to test these transgenics are ROS production (nearly completely dependent on AtrbohD) and a response to pathogen infection to assess the size of the plant, where AtrbohF plays a more important role.

We generated different AtrbohD and AtrbohF alleles, both in vectors suitable to transfer the gene by regular cloning and in a vector that was compatible with the GATEWAY system. The alleles were transferred to binary vectors to allow their constitutive expression under the 35S promoter or under their own promoters, and some transgenic homozygous lines were identified. Intriguingly, the over-expression constructs are not working properly. Most of these lines do not show complementation, suggesting that over-expression of these genes could have deleterious effects on the plant. We also experienced considerable problems in obtaining GFP fused lines in order to monitor the cell localisation of these proteins, obtaining just a few lines that are currently under evaluation. On the other hand, we are obtaining good complementation with constructs with their own promoter, which was obtained by both regular and GATEWAY cloning strategies. We achieved interesting results on the role of the promoter on the specific function of these genes. The analysis of transgenic plants with their promoters fused to the uidA gene revealed that the AtrbohD and AtrbohF promoters are active in several tissues. Interestingly the two promoters are induced by pathogens, although with a different pattern of expression. Additionally, complementation studies with their own promoter indicate the importance of the promoter region to obtain complementation of AtrbohD and AtrbohF mutants.

Turning to the second specific aim, analyses of RBOH functions suggest that ROS act in complex signalling networks which are operational in many pathways in response to developmental cues or to the environment. Particularly relevant are the links between RBOH-dependent ROS production and Ca2+, phosphorylation, and plant regulators like SA and ABA. The response to ozone also induces an oxidative burst that offers similarities to the pathogen-induced oxidative burst, and where RBOH and heterotrimeric G proteins have been implicated (Joo et al., 2005). We wanted to study the interaction between the RBOH and heterotrimeric G proteins in the context of the defence response.

Our analysis of the mutants in the heterotrimeric G proteins reveals that the mutant in the Gß subunit of heterotrimeric G proteins (AGB1) displays enhanced susceptibility to different strains of Pseudomonas syringae, suggesting that this gene is important for basal resistance.

We performed epistasis studies between the G protein mutants and the AtrbohD and AtrbohF mutants. The analysis of the different mutant combinations indicate that heterotrimeric G proteins and NADPH oxidase mediate the same pathway in response to hemibiotrophic bacteria Pseudomonas syringae, whereas they mediate two different pathways in response to the necrotrophic fungus Plectosphaerella cucumerina, stressing the fact that these signals play different functions depending on the typo of pathogen. In addition, epistasis studies between the heterotrimeric G proteins and salicylic acid deficient mutants reveal that the resistance pathway mediated by AGB1 is independent of salicylic acid signalling.

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