WP1. Construction of lines that will facilitate the identification of candidate HR-PCD regulators.
The aim of this WP is the establishment of genetic tools to facilitate identification of potential targets that are relevant to the execution of HR-PCD. The comparison of the two ecotypes Col-0 versus Ws-2 (effective HR-PCD vs. ineffective HR-PCD) represents an excellent paradigm for the study of quantitative and qualitative differences that govern HR-PCD. Furthermore, I will establish another paradigm by knocking-out AtMC genes. The reasoning for that is that atmc1 or atmc4 do not completely abolish HR-PCD, due to the functional redundancy of AtMCs. So far, thorough and comprehensive genetic studies on the AtMC gene family during HR-PCD are limited by their functional redundancy and the clustering on chromosome 1 of six of them (AtMC4-7) which makes difficult the construction of higher order mutants.
We constructed atmc knockouts in Col-0 and Ws-2 backgrounds. To this end, we used three different constructs carrying varying numbers of guide RNA (Deliverable 1) for clustered regulatory interspaced short palindromic repeat (CRISPR-CAS9) targeting. We obtained lines in Col-0 and Ws-2 backgrounds. We identified that only two genes were carrying mutations. Therefore, we are now analysing additional lines.
We set up a protocol for analyses of HR-PCD in leaves of Ws-2 and Col-0 plants. We established that the Pseudomonas fluorescence::avrRPS4 represents an excellent pathosystem for our analyses. Yet, we were not able to assay HR-PCD using ion leakage assay, Infiltrated tissues were used for protein and RNA degradomes and polysomes. Samples have been collected and are now analyzed.
WP2. Unravelling the mRNA degradome, translatome and protein degradome of HR-PCD.
The aim of this WP is to analyze the spatial landscape of mRNA decapping, translatome and proteolysis of RPS4/RRS1-mediated HR-PCD.
We set up a protocol for analyses of HR-PCD in leaves of Ws-2 and Col-0 plants. We found that Ws-2 showed more efficient Pst::avrRPS4 HR-PCD and as suggested here it represents an excellent HR-PCD system. Infiltrated tissues were used for protein and RNA degradomes.
For the protein degradome analysis, we established a new method which facilitates the analyses of a large number of degradomes, with significantly reduced cost.
WP3. Genetics to identify novel key regulators of HR-PCD.
To provide evidence that the previous events are functionally linked to HR-PCD, I will attempt to modify the identified genes in the previous working packages and assess the effect of these modifications on HR-PCD.
In a pilot experiment, we defined a large number of proteolytic events that occurred during HR-PCD induced by avrRPS4, using the setup detailed in WP2. Hence, we screened single mutants of proteases, such as AtMC1, AtMC4 and a triple mutant of Cathepsin B for HR-PCD. We could not find any changes in the rate of avrRPS4 HR-PCD. Therefore, we are now attempting to focus on more proteases that might be relevant to HR-PCD.
