P-180 - Identifying novel virulence genes responsible for blister-like lesions caused by Gram-positive bacterial pathogen Clavibacter michiganensis in Nicotiana benthamiana

The Gram-positive bacterial pathogen Clavibacter michiganensis causes bacterial canker on tomato stem and blisters on leaves. We have shown that this pathogen can cause similar symptoms in Nicotiana benthamiana, including blister-like lesions on leaves. These lesions initially appeared as numerous small white spots. Using DAB and trypan blue staining, the blister-like lesions were the result of cell death and were accompanied by the accumulation of reactive oxygen species. Over time, these lesions developed into necrotic lesions in the leaves. Cryo-SEM observation of blister-like lesions showed that the leaf tissue in the lesions was collapsed and sunken. Notably, a strong GFP signal was detected mainly under the blister-like lesions after infiltration with GFP-tagged C. michiganensis. To identify the genes responsible for the blister-like lesions, a total of 2,148 mutants of C. michiganensis type strain LMG7333^T were infiltrated into N. benthamiana leaves. Among them, 14 mutants with reduced or non-blistering symptoms were selected. Only some of these selected mutants caused reduced wilting symptoms in tomato, suggesting that some genes only play a role in the formation of blister-like lesions. Therefore, our results indicate that blister-like lesions in N. benthamiana leaves are formed due to the cell death caused by accumulation of C. michiganensis bacteria, and that there are some virulence genes that may only be responsible for the formation of blister-like lesions.