Precise CRISPR-Cas9 Mediated Genome Editing in Super Basmati Rice for Resistance Against Bacterial Blight by Targeting the Major Susceptibility Gene

Abstract

Basmati rice is famous around the world for its flavor, aroma, and long grain. Its demand is increasing worldwide, especially in Asia. However, its production is threatened by various problems faced in the fields, resulting in major crop losses. One of the major problems is bacterial blight caused by <i>Xanthomonas oryzae pv. oryzae (Xoo)</i>. <i>Xoo</i> hijacks the host machinery by activating the susceptibility genes (<i>OsSWEET</i> family genes), using its endogenous transcription activator like effectors (TALEs). TALEs have effector binding elements (EBEs) in the promoter region of the <i>OsSWEET</i> genes. Out of six well-known TALEs found to have EBEs in Clade III <i>SWEET</i> genes, four are present in <i>OsSWEET14</i> gene's promoter region. Thus, targeting the promoter of <i>OsSWEET14</i> is very important for creating broad-spectrum resistance. To engineer resistance against bacterial blight, we established CRISPR-Cas9 mediated genome editing in Super Basmati rice by targeting 4 EBEs present in the promoter of <i>OsSWEET14</i>. We were able to obtain four different Super Basmati lines (SB-E1, SB-E2, SB-E3, and SB-E4) having edited EBEs of three TALEs (<i>AvrXa7</i>, <i>PthXo3</i>, and <i>TalF</i>). The edited lines were then evaluated in triplicate for resistance against bacterial blight by choosing one of the locally isolated virulent <i>Xoo</i> strains with <i>AvrXa7</i> and infecting Super Basmati. The lines with deletions in EBE of <i>AvrXa7</i> showed resistance against the <i>Xoo</i> strain. Thus, it was confirmed that edited EBEs provide resistance against their respective TALEs present in <i>Xoo</i> strains. In this study up to 9% editing efficiency was obtained. Our findings showed that CRISPR-Cas9 can be harnessed to generate resistance against bacterial blight in indigenous varieties, against locally prevalent <i>Xoo</i> strains.