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Abstract
Cotton is the most important source of natural fiber with immense agricultural, textile and historic importance. Due to its agricultural importance, it is widely cultivated in monoculture, which increases its vulnerability to many diseases and pests. Bacterial blight of cotton (CBB), caused by Xanthomonas citri pv. malvacearum (Xcm) is the most destructive bacterial disease of cotton worldwide. Development and cultivation of CBB resistant cultivars, as well as the implementation of acid delinting disease management strategies have effectively controlled this disease. Unfortunately, in the recent years, CBB has reemerged in the U.S. due to the increased use of CBB susceptible cultivars. Despite the economic importance of cotton disease management, little is known about fundamental aspects of the CBB disease cycle and Xcm-cotton interactions. The purpose of this work is to elucidate the effect that currently available disease resistance has on Xcm’s initial stages of the disease cycle and to validate novel CBB resistance strategies. We employed an auto-bioluminescently tagged Xcm strain and its Type III secretion system (T3SS-) mutant to investigate the colonization of susceptible and resistant cotton cultivars after seed infection. Through a seed infection assay we found that regardless of cotton cultivar resistance capabilities or the Xcm strain molecular background, cotton cotyledons were always colonized. We also observed that the T3SS contributed to colonization but is not essential in the initial stages of Xcm-cotton host-pathogen interaction. Scanning electron microscopy of cotton cotyledon petioles originating from the infected seedlings showed the presence of Xcm in the xylem, suggesting vascular movement of the pathogen. We investigated the patterns of preferential cotton leaf colonization by Xcm after seedling dip-inoculation. Quantification of free Salicylic acid (SA) and vacuole-stored Salicylic acid beta-glucoside (SAG) did not reveal defects in the SA biosynthesis. Quantification of the SA responsive genes transcripts after benzothiadiazole (BTH) treatment revealed impaired expression of GhPR1 in developing cotton true leaves. We verified that the transient expression of the Elongation Factor Thermo unstable (EF-Tu) receptor from Arabidopsis AtEFR in cotton leads to a gain of elf18Eco recognition, as well as impairment of Xcm colonization in a seed-inoculation assay. We attempted to validate the modified susceptibility gene based resistance strategy in cotton-Xcm host-pathogen interaction using virus induced gene silencing (VIGS) and transient expression of constructed CRISPR Cas9 mediated gene activation and transgene expression. Unfortunately, we did not find any conclusive evidence to support this strategy.