Gene editing can contribute to powdery mildew control strategies

Dr. Laurent Deluc, Associate Professor, Department of Horticulture, Oregon State University. Dr. Satyanarayana Gouthu, Faculty Research Associate, Department of Horticulture, Oregon State University.

 

Powdery mildew (PM), caused by the fungus Erysiphe necator, is a major grapevine disease present in grape-growing areas worldwide. To control this disease, vineyards rely heavily on fungicide applications. However, despite well-planned treatments, powdery mildew infections can still occur. In addition, many commonly used fungicides for powdery mildew control are becoming less effective as the fungus develops resistance, prompting the need for more effective and sustainable treatments.

 

For the past 40 years, there has been a long and interesting story about the relationship between a particular family of genes and various pathogens that cause mildew in many crops. This family of genes is called MLO genes for Mildew locus O. Classified as susceptibility genes, mutations in these genes can confer resistance to PM. The history of the mildew locus (MLO) dates back to 1942, with a report in Barley variants describing the use of X-ray-induced mildew resistance in barley (Freisleben and Lein, 1942). Only in 1997 was the sequence identity of the MLO gene in wild-type barley plants revealed, with the demonstration that MLO gene inactivation can confer resistance to PM in this crop (Büschges et al. 1997). More recently, it has been clearly demonstrated that MLO proteins influence infection outcomes across a broad range of pathogen species, including hemibiotrophs (feed on living tissues) and necrotrophs (feed on dead tissues) fungi (Consonni et al. 2006). Today, MLO-like genes are defined as highly conserved, lineage-specific plant proteins, and, given their ubiquitous presence in modern plant genomes, a core component of plant biology. Based on their protein sequence identity, MLOs have been classified into seven phylogenetic clades or groups (clades I to VII) (Kusch et al. 2016). Most MLOs involved in plant immunity or susceptibility belong to clades IV and V.  More than ~200 published papers on MLOs aimed to achieve engineered resistance to PM by impairing MLO function in various plant species. But there is a caveat: beyond the intended research goal, gene inactivation can sometimes lead to growth and developmental defects because genes have multiple roles in many plant processes, and MLO genes are no exception (Figure 1) (Li and Xiao 2025).

 

mlo genes involved in PM susceptibility have been investigated based on their homology to functionally validated mildew susceptibility genes, AtMLOs 2, 6, and 12, in Arabidopsis Thaliana, also called thale cress, a major genetic model in plant sciences (Consonni et al. 2006). Studies on grapevine have reported the induction of mlo genes during PM infection (Feechan et al. 2009). By either gene silencing or gene editing, VitviMLO13 and 17 seem to be essential for PM susceptibility (Moffa et al. 2025, Pessina et al. 2016). For VitviMLO3 and 4 and their role in PM susceptibility, the scientific evidence was less compelling  (Pessina et al. 2016, Wan et al. 2020), prompting us to clarify the cumulative contribution of clade V MLOs to PM susceptibility.

 

Satyanarayana and I developed a gene-editing project to understand the combinatorial effects of mlo genes on PM susceptibility. The final study is currently under peer review but accessible online (Deluc and Gouthu 2026). Instead of “killing two birds (mlo) with one stone (CRISPR-Cas9), we kill one, two, three, or four birds”. Using the microvine model, we genetically engineered a series of transgenic mutants targeting either one, two, three, or the four mlo genes (simple, double, triple, and quadruple knockouts) to assess the potential combinatorial effects on PM resistance. The grapevine genes VitviMLO3, 4, 13, 17 exhibit a coordinated yet stage-specific expression during PM infection, spanning from early establishment to later phases of fungal growth. Early induction of VitviMLO4 and -17 suggest roles in initial infection, while sustained expression of VitMLO4, 3, and 17 supports later pathogen development. At advanced stages of infection, VitMLO13 and 17 show their primary contribution to PM susceptibility, consistent with a recent report (Moffa et al. 2024). CRISPR-based multiplex editing revealed substantial variability in PM susceptibility, arising from several factors, including the fact that the resulting mutants do not derive from a single cell, as one would expect with the microvine system for genetic engineering. Instead, the genetic fingerprint of a transgenic mutant could derive from several cells that are differentially edited (mosaicism) or not edited at all (a chimeric plant containing a wild-type background).

 

This has made the interpretation of the resistant assays quite challenging. However, we did dissect the functional contributions of the four class-V MLO genes to PM susceptibility across the different mutant types generated. Surprisingly, partial editing of VitvMLO13 and 17 resulted in increased pathogen spread, likely due to mixed populations of susceptible and resistant cells that facilitate fungal expansion. By contrast, the complete disruption of both genes restricted mycelial growth, confirming their central role in promoting infection, as recently shown by Moffa et al. (2025). However, unlike in Moffa et al. (2025), the full double knockout mutant for VitviMLO13 and 17 could not survive transfer to the greenhouse and is maintained only in tissue culture. Satyanarayana also confirmed that VitviMLO3 and 4 specifically contributed to the fungal sporulation stage, highlighting distinct roles among MLO family members beyond initial host penetration. Full knockout of multiple MLO genes often led to severe developmental defects (Wan et al. 2020), indicating that these susceptibility genes also play essential roles in plant growth and physiology. Luckily, one quadrupole-knockout line for the four mlo genes, which was not entirely edited, exhibited strong resistance without many plant growth and physiological penalties (Figure 2), suggesting that residual MLO activity is sufficient to maintain their role in important developmental functions. Overall, the study demonstrated that MLO genes exhibit unequal and partially redundant contributions to PM susceptibility, with VitviMLO17 playing a particularly prominent role, followed in order of importance by VitviMLO13, VitviMLO3, and VitviMLO4 being “the cherry on top”.

 

In conclusion, achieving durable resistance to PM via genetic engineering of mlo genes will require generating mutant lines that reduce PM susceptibility, while preserving at least some MLO activity to maintain normal development. Major efforts should drive the scientific community toward a better understanding of how MLO functions in the context of PM interaction with grapevine. This will likely lead toward implementing a non-GMO, precise gene-editing strategy that targets parts of the MLO candidate proteins critical for conferring PM susceptibility without altering the parts of the MLO proteins that are essential to other developmental functions in grapevine. Another possibility is the development of an RNA interference-mediated silencing approach to reduce the activity of these proteins by topical application of dsRNA molecules, with the objective of reducing the activity of these mlo genes during the growing season to confer PM resistance. The spray application of MLO-derived RNA molecules, aligned with PM infection peaks and integrated into growers' spray programs, could be a potential approach to manage PM in the field in the future, as recently proposed for vineyards (McRae et al. 2023).

 

Additional information

This research project was funded by the American Vineyard Foundation (2019-2021), the Oregon Wine Board (2021-2022), and the PD/GWSS Board (2022 to 2024).

 

Literature Cited

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Li P, Xiao S. 2025. Diverse Functions of Plant MLO Proteins: From Mystery to Elucidation. Annual Review of Phytopathology 63:147–173.

McRae AG, Taneja J, Yee K, Shi X, Haridas S, LaButti K, Singan V, Grigoriev IV, Wildermuth MC. 2023. Spray‐induced gene silencing to identify powdery mildew gene targets and processes for powdery mildew control. Molecular Plant Pathology 24:1168–1183.

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Pessina S, Lenzi L, Perazzolli M, Campa M, Costa LD, Urso S, Valè G, Salamini F, Velasco R, Malnoy M. 2016. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine. Hortic Res 3:1–9.

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