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Regulation of citrus DMR6 via RNA interference and CRISPR/Cas9-mediated gene editing to improve Huanglongbing tolerance

Shujian Zhang: U.S. Horticultural Research Laboratory, USDA-ARS

<div>Plant innate immunity involves the activation of inducible defenses that are tightly controlled by gene regulators. <em>Arabidopsis</em> Down Mildew Resistance 6 (DMR6) is a 2OG Fe (II)-dependent oxygenase that acts as a suppressor of plant immunity, and its expression is upregulated during pathogen infection (Zeilmaker <em>et al</em>., 2015). Mutation of Arabidopsis DMR6 and its orthologs in potato and tomato resulted in elevated salicylic acid level and conferred broad-spectrum disease resistance (de Toledo Thomazella <em>et al</em>., 2016; Sun <em>et al</em>., 2016). Huanglongbing (HLB) is a devastating disease in citrus worldwide. Genetic resistance from commercial varieties is lacking but tolerance has been observed. Previously, we found <em>DMR6-</em>like genes were downregulated in the more tolerant ‘Jackson’ grapefruit compared with those in the susceptible ‘Marsh’ grapefruit. Here, we conducted an expression survey of citrus<em> DMR6</em> orthologs in ‘Hamlin’ sweet orange, ‘Clementine’ mandarin, ‘Carrizo’ citrange, rough lemon, sour orange and citron. The expression levels were significantly higher in infected trees than in uninfected ones regardless genotype, suggesting a universal role of <em>DMR6</em> in the defense against the HLB bacterium,<em> Candidatus </em>Liberibacter asiaticus. RNA interference was used for gene knockdown in Carrizo, and resulted in an average of 41.4% silencing efficiency. <em>DMR6</em> silenced plants exhibited higher expression of defense-related genes than non-transgenic controls. A CRISPR/Cas9 system was also used for gene knockout. Three sgRNAs were designed and shown to be effective in DNA cleavage <em>in vitro</em>. <em>Agrobacterium</em>-mediated transformation of these CRISPR constructs in citrus is underway to generate <em>DMR6</em> mutants for increased HLB tolerance/resistance.</div>