AUTHOR=Tek Mumin Ibrahim , Calis Ozer , Fidan Hakan , Shah Mehraj D. , Celik Sefanur , Wani Shabir Hussain 

TITLE=CRISPR/Cas9 based mlo-mediated resistance against Podosphaera xanthii in cucumber (Cucumis sativus L.)

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.1081506

DOI=10.3389/fpls.2022.1081506

ISSN=1664-462X

ABSTRACT=<p>Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously <italic>mlo</italic>-based resistance was reported in various plants, including cucumber, with generated loss-of <italic>CsaMLO</italic> function mutants. However, <italic>mlo</italic>-based resistance in cucumber is also complex and involves additional mechanisms such as hypersensitive response (HR) and papillae formation. For this reason, we focused on determining the <italic>mlo</italic>-based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for <italic>CsaMLO1</italic>, <italic>CsaMLO8</italic>, and <italic>CsaMLO11</italic> of PM susceptible ADR27 cucumber inbred lines and <italic>CsaMLO</italic> mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect <italic>Podosphaera xanthii</italic> germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated <italic>CsaMLO</italic>s in cucumber are negative regulators in different defense mechanisms against powdery mildew at early and late stages of infection. Further, the experiment results indicated that <italic>CsaMLO8</italic> mutation-based resistance was associated with the pre-invasive response, while <italic>CsaMLO1</italic> and <italic>CsaMLO11</italic> could be negative regulators in the post-invasive defense response in cucumber against <italic>P</italic>. <italic>xanthii</italic>. Although the loss-of <italic>CsaMLO8</italic> function confers the highest penetration resistance, <italic>CsaMLO1</italic> and <italic>CsaMLO11</italic> double mutations could be potential candidates for HR-based resistance against PM pathogen in cucumber. These results highlighted the crucial role of CRISPR/Cas9 to develop PM resistant cucumber cultivars, possessing strong pre-invasive defense with <italic>CsaMLO8</italic> or post-invasive with <italic>CsaMLO1</italic>/<italic>CsaMLO11</italic> mutations.</p>