Different strategies were taken to identify which of the approx. 25 T3SS proteins are recognized by specific wheat cultivars. These include testing if recognition can occur from killed bacteria, and if 14 individual T3SS proteins, some of which were purified, are recognized when injected into the space between the plant cells. The fact that these tests were negative, suggested that the recognition takes place inside the plant cells, and that the recognized protein therefore is one of the proteins transported by the T3SS itself. I therefore, invested considerable efforts in setting up an experimental system for expressing proteins inside wheat cells and scoring their ability to activate the cell death response. I succeeded and until now, I have expressed those nine of the approx. 25 proteins, which are most likely to be transported by the T3SS, inside the cells of wheat. However, my efforts have been in vain, as none of them activated cell death.
In order to identify the wheat protein that functions as receptor for the T3SS, I studied the seven genes, genetically defined to encode candidate receptors. Among those, I concluded a plasma membrane receptor kinase (PMK) and three wall-associated kinases (WAK) to be the obvious candidates. In collaboration with the Chinese Academy of Science, Beijing, knock-out mutants were made in the PMK gene, while this turned out not to be possible in the WAK genes. Studies of the pmk mutant have been delayed by the Covid-19.
I have provided evidence that the wheat responses activated by the T3SS are classical immune responses, and therefore likely to protect the plant against bacterial attack. To test whether this is the case, I inoculated the wheat plants with a Xanthomonas pathogen, but observed no effect on disease development in cultivars able to recognize T3SS. The reason for this result could be a molecular difference between the T3SSs from Xanthomonads and Pseudomonads. I have in the meantime obtained a wheat Pseudomonas pathogen, which I will test in the coming months.
