FaNPR3 Members of the NPR1-like Gene Family Negatively Modulate Strawberry Fruit Resistance against <i>Colletotrichum acutatum</i>

Strawberry fruit is highly appreciated worldwide for its organoleptic and healthy properties. However, this plant is attacked by many pathogenic fungi, which significantly affect fruit production and quality at pre- and post-harvest stages, making chemical applications the most effective but undesirable strategy to control diseases that has been found so far. Alternatively, genetic manipulation, employing plant key genes involved in defense, such as members of the NPR-like gene family, has been successful in many crops to improve resistance. The identification and use of the endogenous counterpart genes in the plant of interest (as it is the case of strawberry) is desirable as it would increase the favorable outcome and requires prior knowledge of their defense-related function. Using RNAi technology in strawberry, transient silencing of <i>Fragaria ananassa</i> NPR3 members in fruit significantly reduced tissue damage after <i>Colletotrichum acutatum</i> infection, whereas the ectopic expression of either <i>FaNPR3.1</i> or <i>FaNPR3.2</i> did not have an apparent effect. Furthermore, the ectopic expression of <i>FaNPR3.2</i> in <i>Arabidopsis thaliana</i> double-mutant <i>npr3npr4</i> reverted the disease resistance phenotype to <i>Pseudomonas syringe</i> to wild-type levels. Therefore, the results revealed that members of the strawberry FaNPR3 clade negatively regulate the defense response to pathogens, as do their Arabidopsis AtNPR3/AtNPR4 orthologs. Also, evidence was found showing that FaNPR3 members act in strawberry (<i>F. ananassa</i>) as positive regulators of <i>WRKY</i> genes, <i>FaWRKY19</i> and <i>FaWRKY24</i>; additionally, in Arabidopsis, FaNPR3.2 negatively regulates its orthologous genes <i>AtNPR3/AtNPR4</i>. We report for the first time the functional characterization of FaNPR3 members in <i>F. ananassa</i>, which provides a relevant molecular basis for the improvement of resistance in this species through new breeding technologies.