Periodic Reporting for period 1 - T-REX (Developing healthy crops for EU: T3SS-Recognition EXploration (T-REX) for plant immunity against bacteria) Reporting period: 2019-01-14 to 2021-01-13 Summary of the context and overall objectives of the project Plants, animals and humans often suffer from bacterial diseases, which seriously affect production and well-being. Pathogenic bacteria use the needle-like type-3 secretion system (T3SS) to inject effector proteins into the cells of the hosts. At the same time, it is important for the hosts to be able to recognize invading pathogens, in order to activate their immune system when attacked. Therefore, it is surprising that it has not previously been demonstrated that plants can recognize the T3SS, as it is indispensable for bacterial pathogens. Meanwhile, this project was based on our finding that many wheat cultivars indeed do recognize the T3SS from a Pseudomonas pathogen, and that the wheat gene responsible for this should be found between seven candidates. The objectives of the project were 1) to identify which of the approx. 25 T3SS proteins is recognized by wheat, 2) to identify which wheat protein functions as receptor to detect this T3SS protein, and 3) to evaluate whether the recognition activates immunity that protects the plant. Understanding the molecular details of this recognition can potentially be exploited to control serious bacterial diseases in plants and animals. Such strategies include use of selected naturally occurring gene variants, molecularly modified genes or drugs inspired by the structures of the interacting molecules. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) I will continue my studies in the rest of 2021 based on other funding. Here I aim to further refine my tests of T3SS proteins in the wheat cell expression system, re-test those that I have tested already, and analyze more candidates between the approx. 25 proteins. Secondly, I hope to be able to demonstrate PMK to be the T3SS receptor through studies of the generated pmk mutant. Thirdly, I will test the disease progression of the recently obtained Pseudomonas bacteria, to see if the T3SS recognition confers disease resistance. Finally, I will write a scientific paper, describing our discoveries. These will include the fundamental genetic studies of the T3SS recognition in wheat and the findings in this Marie Skłodowska-Curie Individual Fellowships.