Reverse Genetic Screening Identifies Genes Involved in the Pseudomonas simiae WCS417 Drought Stress Rescue of Arabidopsis thaliana

Drought stress in plants threatens global food security, stunting the production of staple food crops and drastically lowering crop yields. Beneficial interactions between plants and rhizosphere-inhabiting microbes can serve as biological solutions to environmental stressors such as drought, allowing for recovery of plant productivity, growth, and development. Some plant growth promoting rhizobacteria (PGPR) are known to improve the fitness of certain plants under various forms of abiotic stress, but drought stress rescue remains poorly studied and understood. In this experiment, we employed the well-characterized PGPR&nbsp;<em>Pseudomonas simiae</em>&nbsp;WCS417 to identify the specific genes and mechanisms by which the PGPR can confer drought stress rescue in the model plant&nbsp;<em>Arabidopsis thaliana</em>. After verifying the partial rescue of&nbsp;<em>A. thaliana</em>&nbsp;by WCS417 under drought conditions, we employed a reverse genetic screen of 48 Tn5-transposon insertion mutants of WCS417, selected from literature. The screen identified two candidate genes,&nbsp;<em>ilvB</em>&nbsp;and&nbsp;<em>ilvC</em>, that displayed a complete loss-of-rescue phenotype in&nbsp;<em>A. thaliana&nbsp;</em>when gene function was disrupted. Both genes are involved in the biosynthesis of branched chain amino acids (BCAA), and the mutants displayed decreased colonization of the plant, suggesting that BCAAs may play a role in the bacterial fitness and colonization efficiency of WCS417. Maintaining good bacterial fitness under stress may be essential to the utilization of bacterial biosolutions in agriculture, and understanding the mechanisms of drought stress rescue by rhizobacteria will pave the way to successful applications of microbial biostimulants in the field.