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

Abstract

Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously mlo-based resistance was reported in various plants, including cucumber, with generated loss-of CsaMLO function mutants. However, mlo-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 mlo-based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for CsaMLO1, CsaMLO8, and CsaMLO11 of PM susceptible ADR27 cucumber inbred lines and CsaMLO mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect Podosphaera xanthii germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated CsaMLOs 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 CsaMLO8 mutation-based resistance was associated with the pre-invasive response, while CsaMLO1 and CsaMLO11 could be negative regulators in the post-invasive defense response in cucumber against P. xanthii. Although the loss-of CsaMLO8 function confers the highest penetration resistance, CsaMLO1 and CsaMLO11 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 CsaMLO8 or post-invasive with CsaMLO1/CsaMLO11 mutations.

CsMLO8/11 are required for full susceptibility of cucumber stem to powdery mildew and interact with CsCRK2 and CsRbohD

“…PM resistance in the adult and young leaf was also evaluated. Consistent with data from Tek et al [ 41 ], the PM infection on mlo8 and mlo11 leaves was markedly decreased compared with the WT. Moreover, the PM symptoms and spore density decreased more noticeably on the stem and hypocotyls compared with the leaves, indicating that disruption of CsMLO8 and/or CsMLO11 results in greater reduction of PM infection on stems than on leaves.…”

“…[ 49 ] recently screened a PM-susceptible mutant from an EMS-mutagenized population of a natural mlo8 mutant, and also found that PM resistance of mlo8 was associated with increased cell wall deposition, while in mlo11 CR1 and mlo8 CR1 mlo11 CR1 defense response pathways were significantly upregulated, suggesting that CsMLO8 and CsMLO11 might have different defense mechanisms. Our findings are supported by the study of Tek et al [ 41 ], in which CsMLO8 was proposed as a negative regulator in the pre-invasive response, while CsMLO11 could be associated with the post-invasive defense response.…”

“…Later studies showed that the overexpression of CsMLO1 in the tomato mlo mutant completely restored PM susceptibility, while overexpression of CsMLO11 only led to partial restoration [ 33 ]. CsMLO1 , CsMLO8 , and CsMLO11 mutants were recently generated in cucumber plants [ 41 ]; the PM symptoms on mlo8mlo11 and mlo1mlo11 leaves were significantly decreased compared with mlo1 plants, and no PM symptoms appeared on mlo1mlo8mlo11 leaves [ 41 ]. However, PM resistance on stem or hypocotyl was not characterized in these studies.…”

From Sequencing to Genome Editing in Cucurbitaceae: Application of Modern Genomic Techniques to Enhance Plant Traits

“…Modern gene-editing techniques, however, can elucidate the role of individual genes, which can be crucial for effective resistance breeding. Tek et al [126] used CRISPR/Cas9 to investigate the function of genes responsible for mlo resistance to powdery mildew. A mutation in CsaMLO8 corresponded to pre-invasion response resistance, while genes CsaMLO1 and CsaMLO11 were identified as negative regulators in a post-invasive response to Podosphaera xanthii (Table 2).…”

NLR- and mlo-Based Resistance Mechanisms against Powdery Mildew in Cannabis sativa

“…Knocking-out the MLO ortholog SlMLO1 resulted in full resistance to the PM fungus in tomatoes [106]. CRISPR/Cas9-mediated mutagenesis of the MLO3 gene provided an enhanced resistance to PM in grapevines [107], MLO7 was used as a host susceptibility gene to improve grapevine and apple disease resistance to PM [108] and mlomediated resistance against Podosphaera xanthii was successfully used in cucumber [109]. Furthermore, a targeted deletion in the wheat MLO-B1 locus conferred robust PM without growth penalty and yields loss [110] These findings clearly show that the manipulation of S genes, such as MLO genes, is a powerful approach to generate pathogen resistance in important crops.…”