From the 475 predicted Hpa effectors (containing RxLR, RxLL and RxLCRN motifs), I selected 77 with high expression at early stage of the infection for synthesis. These were cloned as GFP fusion proteins via modular Golden Gate cloning in to plant expression vectors to examine their subcellular localization in N. benthamiana leaves. I developed a ‘2-in-1’ expression system to quantify the mobility of effector proteins in vivo and quantified the mobility of 19 effectors. I identified 9 effectors with greater than expected (‘super-mobile’) mobility effectors. I selected the 5 effectors with higher expression levels during infection for further analysis. This screen has established a comprehensive catalogue of Hpa effector localisations and cell-to-cell mobilities
Hpa is not transformable. Thus, to validate the role in virulence of our selected super-mobile effectors, we have generated transgenic Arabidopsis lines that overexpress these effectors with the aim to examine their impact on Hpa infection. To reduce any effect of the effector on development of the host, we have also generated inducible promoter (estradiol) lines for effector expression. To knockdown the effectors, we aim to use a HIGS system. We have generated host transgenic lines carrying dsRNA constructs which will allow us to examine if these lines are more resistant to Hpa. These tools are made and in the final stages of selection for homozygous lines suitable for virulence assays.
To investigate how host gene expression changes are affected by symplastic connectivity, we used RNAseq to compare transcriptional changes in lines in which we can manipulate PD aperture. Initially, we assayed two PD-closed lines: DMR6-cals3m and Est-PLUG. In contrast to our expectations, both lines were more susceptible to Hpa. Therefore, we changed our strategy and used the pdlp1,2,3 mutant which is more susceptible to Hpa and does not induce callose deposition in the walls of infected cells. We performed RNAseq on these mutants and were surprised to see that there were greater differences between mutants and Col-0 during development than during infection. However, we have identified and validated the differential regulation of the phosphate transporter PHT1;1 which raises new hypotheses about nutrient distribution when the symplast is mis-regulated.
To investigate infection-triggered activation of sucrose transporters, we generated a SUC2pro:GUS construct and assayed for expression in N.benthamiana leaves in the presence and absence of different cell-to-cell mobile effectors. Preliminary data showed that most effectors induced SUC2 expression and therefore there are likely effects of the transient expression system. I decided this objective would be best pursued in stable transgenic plants.
