Clade V MLO proteins are bona fide host susceptibility factors required for powdery mildew pathogenesis in Arabidopsis

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Clade V MLO proteins are bona fide host susceptibility factors required for powdery mildew pathogenesis in Arabidopsis David Bloodgood , View ORCID Profile Qiong Zhang , View ORCID Profile Pai Li , Ying Wu , Michael Pan , Christina Zhou , Apsen Hsu , Jun Zhang , Ralph Panstruga , Sharon Kessler , Ping He , Libo Shan , Chang-I Wei , View ORCID Profile Shunyuan Xiao doi: https://doi.org/10.1101/2025.06.18.660284 David Bloodgood 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qiong Zhang 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Qiong Zhang Pai Li 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pai Li Ying Wu 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael Pan 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christina Zhou 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Apsen Hsu 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jun Zhang 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ralph Panstruga 2 Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University , Worringerweg 1, 52056 Aachen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sharon Kessler 3 Department of Botany and Plant Pathology, Purdue University , West Lafayette, Indiana 47907 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ping He 4 Departmentof Molecular, Cellular, and Developmental Biology, University of Michigan , Ann Arbor MI 48109 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Libo Shan 4 Departmentof Molecular, Cellular, and Developmental Biology, University of Michigan , Ann Arbor MI 48109 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chang-I Wei 5 Department of Nutrition and Food Science, University of Maryland College Park , MD 20742 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shunyuan Xiao 1 Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, MD 20850 6 Department of Plant Sciences and Landscape Architecture, University of Maryland College Park , MD 20742 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shunyuan Xiao For correspondence: xiao{at}umd.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Obligate biotrophic powdery mildew (PM) fungi strictly require living host to survive. To search for host factors or processes essential for PM pathogenesis, a tailored genetic screen was conducted with the immuno-compromised eds1-2/pad4-1/sid2-2 ( eps ) triple Arabidopsis mutant. This led to the identification of five allelic disruptive mutations in Mildew Locus O 2 ( MLO2 ) to be responsible for the compromised-immunity-yet-poor infection (cipi) mutant phenotype upon challenge from an adapted PM isolate. Moreover, the eds1/pad4/sid2/mlo2/mlo6/mlo12 ( eps3m ) sextuple mutant display near complete immunity to the adapted PM fungus without sign of defense activation, demonstrating that these three clade V MLOs in Arabidopsis are bona fide host susceptibility factors of PM fungi. Confocal imaging revealed focal accumulation of MLO2-GFP in the peri-penetration peg membranous space, implicating MLO2 in repairing and stabilizing the damaged host plasma membrane, which may be co-opted by PM fungi for haustorium differentiation. Results from domain-swapping analysis between MLO1 and MLO2 suggest a bipartite functional configuration for MLO2: its C-terminus determines where and when MLO2 functions, while its N-terminal seven transmembrane domain region executes the cellular function that is critical for PM pathogenesis. Genetic studies further demonstrate that, unlike MLO7 in synergids, focal accumulation of MLO2 does not depend on FERONIA (FER) and its five other family members, nor does it require phosphatidylinositol 4,5-bisphosphate produced from phosphatidylinositol 4-phosphate 5-kinase 1 (PIP5K1) and PIP5K2. Together, these findings define clade V MLOs as host factors co-opted by obligate biotrophic PM fungi for successful host colonization. Introduction Powdery mildew (PM) fungi are obligate biotrophic pathogens that strictly require living host cells to survive and thrive ( Schulze-Lefert and Panstruga, 2003 ). Upon landing on a host leaf surface, a PM spore germinates within six hours, forming an appressorium to penetrate the host cell wall within 7–10 hours. The sporeling then differentiates the haustorium physically inside the host cell from the tip of the penetration peg in 12-14 hours ( Koh et al., 2005 ). Concomitant with the development of the haustorium, a host-derived extra-haustorial membrane (EHM) is formed to encase the haustorium ( Wang et al., 2009 ; Berkey et al., 2017 ). The haustorium is believed to mature in 20-24 hours and then it is capable of extracting water and nutrient from the host cell to support hyphal growth on the leaf surface. Within four to five days, the fungus develops an extensive mycelial network, capable of forming conidiophores that produce new conidia, thereby completing its asexual life cycle. Like other obligate biotrophic pathogens, PM fungi cannot be cultured and are genetically intractable. Despite extensive studies into plant-PM fungal interactions, a critical question regarding PM biotrophy remains unresolved: aside from host-derived nutrients, are there any specific host factors or processes that are truly indispensable for PM pathogenesis? Conventional genetic screens have been conducted to identify mutants that show enhanced resistance to PM fungi. Many such mutants show lesion mimic or autoimmunity, with disruptive mutations in likely negative regulators of immunity ( Frye and Innes, 1998 ; Tang et al., 2005 , 2006 ; Wang et al., 2008 ; Zhang et al., 2008 ). To identify possible host factors or processes that are critical for PM pathogenesis without the complication of autoimmunity, we constructed a triple mutant in which genes encoding two essential immune signaling components, EDS1 ( Falk et al., 1999 ) and PAD4 ( Jirage et al., 1999 ), and a key enzyme, SID2, required for salicylic acid (SA) biosynthesis ( Wildermuth et al., 2001 ) were mutated. This eds1-2/pad4-1/sid2-2 ( eps ) mutant is super-susceptible to the adapted PM isolate Golovinomyces cichoracearum ( Gc ) UCSC1 ( Zhang et al., 2018 ). We then performed a large-scale genetic screen using EMS-mutagenized seeds of eps to identify mutants that show c ompromised i mmunity yet p oor i nfection ( cipi ), with the goal of identifying potential host susceptibility factors required for PM pathogenesis. Among the 18 cipi mutants isolated, five mutants with the poorest infection each contains a causal mutation in MLO2 (At1g11310) (see the Results section), suggesting that mlo2 -conditioned poor infection can be uncoupled from defense activation. The MILDEW LOCUS O (MLO) was originally discovered to confer broad-spectrum resistance to PM fungal isolates in barley ( Hordeum vulgare; Hv ) and recessive mutations in the HvMLO gene encoding a seven transmembrane-domain (7TM) protein are responsible for the resistance ( Buschges et al., 1997 ). HvMLO belongs to a plant lineage-specific 7TM protein family ( Kusch et al., 2016 ). Impairment of Arabidopsis thaliana ( At ) MLO2 , MLO6 and MLO12 also results in near complete resistance to an adapted PM isolate ( Consonni et al., 2006 ). These earlier findings stimulated targeted mutagenesis of MLO genes as a promising new avenue for creating PM-resistant crops in recent years ( Feechan et al., 2008 ; Pavan et al., 2011 ; Zheng et al., 2013 ; Wang et al., 2014 ; Kusch and Panstruga, 2017 ; Nekrasov et al., 2017 ; Wan et al., 2020 ). Unfortunately, mlo mutants with strong or near-complete resistance to PM fungi often display reduced plant stature, elevated levels of SA, increased callose deposition, and early leaf senescence, resembling characteristics of autoimmunity ( Wolter et al., 1993 ; Peterhansel et al., 1997 ; Piffanelli et al., 2002 ; Consonni et al., 2006 ; Humphry et al., 2006 ; Kusch et al., 2019 ). Thus, MLOs have been thought to be host susceptibility factors for PM fungi and act as negative regulators of plant immunity, leading to the inference that mlo -mediated resistance is attributable to constitutive or elevated immune responses ( Buschges et al., 1997 ; Acevedo-Garcia et al., 2014 ). Intriguingly, no genetic components known to function in classical pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or effector-triggered immunity (ETI) have been shown to be essential for mlo -mediated resistance in Arabidopsis ( Consonni et al., 2006 ; Miklis et al., 2007 ; Kuhn et al., 2017 ; Kusch et al., 2019 ). These observations suggest two possibilities: either MLO proteins repress a potent, yet uncharacterized defense pathway or MLOs are required for a host cellular process critical for PM fungal pathogenesis—such that the observed autoimmunity in mlo mutants is a downstream consequence of MLO loss, rather than the primary cause of pathogen resistance. MLO genes in plants belong to a small-to-medium sized gene family with varying members (from a few to a few dozens) that can be divided into seven clades based on protein sequences ( Kusch et al., 2016 ). The genome of Arabidopsis thaliana contains 15 MLO family members, falling into five clades. Simultaneous impairment of the three clade-V members, MLO2 , MLO6 and MLO12 , results in near-complete resistance to adapted PM pathogens ( Consonni et al., 2006 ). All MLO genes whose functional impairment confers PM resistance in dicots belong to clade V, whereas in monocots, such resistance-associated MLO s fall within clade IV (Reviewed by Li and Xiao, 2025 ). The functional equivalence of these two clades is suggested by the absence of clade V MLO s in monocots ( Kusch et al., 2016 ). Interestingly, mutations in barley HvMLO1 (clade IV) or wheat TaMLO1 (clade IV), or Medicago MtMLO8 (clade V) cause a reduction in colonization by the arbuscular mycorrhizal fungus Rhizophagus irregularis in their respective mutant hosts ( Jacott et al., 2020 ). This suggests that clade IV/V MLOs serve a conserved and important role in accommodating host entry of biotrophic fungi regardless of whether the interaction is mutualistic or pathogenic ( Jacott et al., 2021 ). Several other Arabidopsis MLO genes have been shown to be involved in distinct biological processes, including root gravitropism and thigmomorphogenesis ( MLO4 and MLO11 ) ( Chen et al., 2009 ; Bidzinski et al., 2014 ; Zhu et al., 2021 ), pollen tube development and guidance ( MLO5 , MLO9 , MLO10 , MLO15 ) ( Meng et al., 2020 ; Zhang et al., 2020 ), and pollen tube reception involving synergid cells ( MLO7 , also known as NOTIA ) ( Kessler et al., 2010 ; Jones et al., 2017 ). All predicted MLO proteins are characterized by an N-terminal region containing seven transmembrane (7-TM) domains and a cytosolic C-terminal tail. This C-terminal region harbors a calmodulin-binding domain (CaMBD), which interacts with calmodulin (CaM) or CaM-like (CML) proteins ( Kim et al., 2002 ; von Bongartz et al., 2023 ). Several studies have shown that calmodulin binding and/or calcium signaling is required for the full functions of distinct MLOs ( Kim et al., 2002 ; Meng et al., 2020 ; Zhu et al., 2021 ; Yuan et al., 2025 ). Excitingly, several Arabidopsis MLO proteins including MLO2 have been shown to possess calcium channel activity when expressed in animal cells ( Cui et al., 2022 ; Gao et al., 2023 ), suggesting that MLOs may also play conserved roles in calcium-homeostasis and calcium signaling in plant cells. However, how MLOs’ calcium channel activity is associated with their distinct biological functions in different cells/tissues or subcellular compartments remain unclear. A GFP-tagged version of barley MLO (HvMLO-GFP) was shown to accumulate focally at the site of cell wall penetration by Blumeria graminis f. sp. hordei ( Bgh ) in barley epidermal cells ( Bhat et al., 2005 ). This observation led to speculation that HvMLO is actively recruited by the fungus to modulate vesicle-associated processes at the plant cell periphery, thereby facilitating host entry by Bgh ( Panstruga, 2005 ). Interestingly, in Arabidopsis, MLO7-GFP is expressed in the synergids of the female gametophyte and is re-localized from Golgi bodies to the filiform apparatus of synergid cells upon arrival of the pollen tube, which is reminiscent of HvMLO’s focal accumulation at the fungal penetration site. Moreover, expressing MLO2-GFP from the MLO7 native promoter largely restored fertility of the mlo7 mutant, whereas MLO1-GFP failed to do so ( Jones et al., 2017 ). Interestingly, the chimeric protein NTA-MLO1 CTerm (also named faNTA) resulting from swapping the C-terminal CaMBD of MLO1 with that of MLO7 was found to be localized to the filiform apparatus of the synergids, similarly as MLO1, and it fully restored fertility of mlo7 ( Ju et al., 2021 ). This suggests that the C-terminus of MLO7 specifies its Golgi® filiform apparatus trafficking upon pollen tube arrival ( Ju et al., 2021 ), which may be regulated through binding of cognate CaM proteins ( Yuan et al., 2025 ). More recently, MLO6 has been shown to interact and colocalize with EXO70H4, a subunit of the exocyst complex, in trichome cells. These two proteins each depends on the other for correct localization and both are required for callose deposition in the trichome cell wall ( Huebbers et al., 2024 ). These findings suggest that MLO6 and likely its two close homologs, MLO2 and MLO12, play an important role in exocytosis of cell wall components ( Huebbers et al., 2024 ). In this study, we show that loss-of-function mutations in MLO2 , MLO6 , and MLO12 result in a near-complete failure of PM pathogenesis, even in eds1/pad4/sid2 triple and eds1/pad4/sid2/pen1/pen2/pen3 sextuple mutant backgrounds, both of which are severely compromised in immune signaling and defense against various (potential) pathogens. These observations suggest that the failure of PM infection in mlo2/mlo6/mlo12 mutants is not immunity driven. Instead, it likely reflects disruption of a host cellular process essential for PM fungal invasion, which may be co-opted by the pathogen during pathogenesis. We further show that MLO2’s focal accumulation around the penetration peg is determined by its CaMBD-containing C-terminus and that Feronia (FEN) and phosphatidylinositol 4-phosphate 5-kinase 1 (PIP5K1) and PIP5K2 are dispensable for MLO2’s focal accumulation and function that is co-opted by PM fungi for host entry and colonization. Results Identification of five allelic MLO2 mutations conferring “resistance” to powdery mildew independent of EDS1, PAD4 and SID2 Plants of the eps mutant are super-susceptible to the adapted PM isolate Golovinomyces cichoracearum (Gc ) UCSC1 ( Fig. 1A ) ( Zhang et al., 2018 ). To identify host genes crucial for biotrophic pathogenesis of PM fungi, we designed and conducted a large-scale mutant screen in the background of the eds1/pad4/sid2 (eps) triple mutant. We aimed to isolate higher-order mutants with compromised immunity yet poor infection (cipi) phenotypes. We reasoned that such cipi mutations would more likely disrupt genes essential for PM pathogenesis independent of immunity activation. Eighteen cipi mutants were isolated, of which five ( cipi2, cipi3, cipi11, cipi12, and cipi15 ) showed similar poorest infection by Gc UCSC1 ( Fig. 1A ; Supplementary Fig. S1A ). Spore quantification showed that sporulation of Gc UCSC1 in cipi3 plants was reduced to only ∼16% of that in eps plants, which is only 1/3 of that in Col-0 ( Supplementary Fig. S1B ). Interestingly, we noticed that trichomes of those five cipi mutants tend to support more fungal growth and sporulation ( Fig. 1B ). To determine if the causal mutations in the five cipi mutants occur in the same gene, we made four crosses between these cipi mutants and found that all F 1 plants showed similar poor infection by Gc UCSC1 ( Fig. 1C ). This result indicates that the causal mutations in the five mutants are allelic. Microscopic examination of PM-infected leaves revealed greatly reduced haustorium formation and conidiophore development, except for occasionally infected trichomes in cipi3 ( Fig. 1D ) and other allelic cipi mutants. We then crossed cipi2 with the eps parental line and derived an F 2 segregating population for genetic mapping of the cipi2 causal mutation. Whole genome sequencing of the bulked segregant pool consisting of 65 F 2 individuals with cipi2 -like phenotype revealed a C-to-T synonymous substitution in MLO2 (At1g11310) in the cipi2 mutant to be co-segregating with the cipi phenotype. This mutation is predicted to create an exonic cryptic donor splice site in the 10th exon of MLO2 ( Fig. 2A ), which is predicted to result in a 47 nt deletion of the 10th exon starting from the mutation site, generating a premature stop codon in the 12th exon ( Fig. 2A,B ). Using the same strategy, a G-to-A synonymous mutation in occurred in MLO2 of cipi3 in the acceptor splice site in the 2nd intron ( Fig. 2A ). This substitution is predicted to activate an immediate downstream exonic cryptic acceptor splice site in the beginning of the 3rd exon, which in turn causes a frameshift resulting in a premature stop codon ( Fig. 2C ). To check if the transcripts of MLO2 from these two mutants are indeed mis-spliced, we performed RT-PCR using total RNA extracted from cipi2 and cipi3 and found that the transcripts were indeed mis-spliced from cipi2 or cipi3 as predicted ( Supplementary Fig. S2 ). The aberrant transcripts are predicted to produce truncated MLO2 proteins of 380 amino acids ( cipi2 ) or 97 amino acids ( cipi3 ) (Fig, 2D,E). These truncated proteins are probably either nonfunctional or not made due to nonsense-mediated mRNA decay ( Baker and Parker, 2004 ). Next, we sequenced the MLO2 genomic DNA in cipi11 , cipi12 and cipi15 , and found that each of the three mutants contains a single nonsynonymous exonic mutation in MLO2 . As depicted in Fig. 2F , cipi11 contains a G-to-A mutation that changes glycine to arginine (G66R) in the second transmembrane domain, which is identical to the previously reported mlo2-8 allele ( Consonni et al., 2006 ). cipi12 contains a G-to-A mutation resulting in a glutamic acid to lysine exchange (E7K) in the N-terminal extracellular domain, which has not been reported before. cipi15 harbors a G-to-A mutation resulting in an aspartic acid to asparagine substitution (D287N) in the second cytoplasmic loop, which is identical to the mlo2-11 allele reported before ( Consonni et al., 2006 ). Download figure Open in new tab Figure 1. Isolation and phenotypic characterization of five allelic cipi mutants. Plants were inoculated with an adapted PM isolate Golovinomyces cichoracearum ( Gc ) UCSC1. Photos of infected plants or leaves were taken at 10-15 dpi. (A) Representative plants of the five cipi mutants along with Col-0 and the eps parental line showing their infection phenotypes at 11 dpi. (B) Representative leaves from eps and cipi3 at 13 dpi. Note the trichome-based infection in cipi3 . (C) Infection phenotypes of F1 hybrid plants derived from crosses between the indicated mutants at 12 dpi. (D) Microscopic images showing fungal structures stained by trypan blue on the leaf surface (upper panel) or inside the epidermal cells (lower panel) of the indicated genotypes at 11 dpi. Note that the trichome cells supporting sporulation in the cipi3 mutant. Arrowheads indicate rod-shaped conidiophores. Arrows indicate haustoria. Dashed lines highlight a trichome cell. Bar=100 μm. Download figure Open in new tab Figure 2. Identification of the five allelic causal mutations in MLO2 . Bulked segregant pool-based whole genome sequencing identified the candidate causal mutations in cipi2 and cipi3 to be in MLO2 . Targeted sequencing of MLO2 in cipi11 , cipi12 and cipi15 revealed disruptive mutations as their respective candidate causal mutations. (A) Schematic of the MLO2 gene structure showing the positions of the nucleotide substitutions in the five cipi mutants. “E” indicates exon. (B-C) Schematic illustration of the positions of the cryptic splice sites (underlined) and the resulting premature stop codons (marked by red hexagons) caused by the mutations in cipi2 (B) and cipi3 (C). (D-F) Topological illustration of the deduced MLO2 mutant proteins resulting from the cipi2 (D), cipi3 (E), and the remaining three cipi mutations (F). Highlighted in red dots are three amino acid substitutions in cipi11 , cipi12 , and cipi15 (F). Blue horizontal bars represent membrane bilayers and gray vertical bars represent transmembrane domains. (G) Representative plants from the indicated genotypes infected with Gc UCSC1 at 12 dpi. Numbers in white circles indicate the number of T1 transgenic plants showing that infection phenotype. To provide further genetic evidence that mutations in MLO2 are responsible for the poor infection phenotype of the five cipi mutants, we generated transgenic cipi3 plants expressing MLO2-GFP from the native MLO2 promoter. Half of the 24 T1 cipi3 plants transgenic for pMLO2:MLO2-GFP restored susceptibility to Gc UCSC1 to a level close to that of eps ( Fig. 2H ) and the remaining 12 showed medium to low levels of susceptibility. These results further demonstrates that the poor PM infection phenotypes in cipi3 and other four cipi mutants were indeed caused by functional impairment of MLO2 and that the MLO2-GFP fusion protein is probably fully functional. Taken together, these genetic data demonstrate that loss of MLO2 significantly reduces PM infection in the eps triple mutant background, mirroring the enhanced resistance observed in mlo2 mutants of Col-0 ( Vogel and Somerville, 2000 ; Consonni et al., 2006 ). These findings further indicate that the reduced PM infection is independent of EDS1, PAD4, and SID2-mediated immune signaling. Loss of MLO2 , MLO6 and MLO12 blocks PM pathogenesis in the eds1/pad4/sid2 background The mlo2-5/mlo6-2/mlo12-1 triple mutants in the Col-0 background (designated 3m/C ) exhibited near complete resistance to PM ( Consonni et al., 2006 ). To see if 3m -mediated resistance can be recapitulated in the eps background, we knocked out MLO6 and MLO12 in cipi3 by CRISPR to create three eds1/pad4/sid2/mlo2/mlo6/mlo12 ( eps3m ) mutants with indel mutations resulting in early stop codons in MLO6 and MLO12 ( Supplementary Fig. S3 ). Infection tests with Gc UCSC1 showed that plants of these three sextuple mutants support no visible fungal growth ( Fig. 3A ). Microscopic examination revealed invariable arrest of PM sporelings shortly after development of the appressorium (arrow in Fig. 3B ) and no haustorium formation in the pavement cells ( Supplementary Fig. S4 ). This suggests that the fungus fails to differentiate haustoria from tip of the penetration peg underneath of the appressorium in pavement epidermal cells. Intriguingly, haustorium formation and limited hyphal development could occasionally be found in trichomes (Inset in supplementary Fig. S4), albeit hardly visible to the naked eye, suggesting that the trichome cell environment is different from that of pavement cells. The uninfected or infected plants of the eps3m mutants did not show early leaf senescence reported for the 3m/C triple mutant ( Consonni et al., 2006 ), nor did they exhibited any other obvious developmental phenotypes ( Fig. 4A ), suggesting that the near-complete resistance to Gc UCSC1 is unlikely due to activation of host defense programs. Download figure Open in new tab Figure 3. Loss of MLO2/6/12 in eps results in complete lack of infection by powdery mildew. An eds1-2/pad4-1/sid2-2 mlo2/mlo6/mlo12 sextuple mutant line ( eps3m-7 ) was created using CRISPR targeted mutagenesis. Plants of this line were subjected to infection tests and transformation. (A-B) Plants of the indicated genotypes inoculated with Gc UCSC1. Plant photos (A) or micrographs after trypan blue staining (B) were acquired at 10 dpi or 6 dpi with Gc UCSC1, respectively. The arrow indicates penetration peg developed from the appressorium. Bars=50 μm. (C) Infection phenotypes of plants of the indicated genotypes inoculated with a virulent oomycete isolate Hyaloperonospora arabidopsidis Noco2. Photos were taken at 7 dpi. (D) Gc UCSC1-infection phenotypes of eps3m and one eps3m transgenic line expressing MLO2-GFP from the MLO2 promoter. Photos were taken at 10 dpi. Download figure Open in new tab Figure 4. mlo -conditioned early leaf senescence is fully suppressed in eps and mlo -mediated “immunity” is not associated with cell death. Plants of Col-0, mlo2-5;mlo6-2;mlo12-1 ( 3m/ Col-0), eps and eps3m were grown under short-day for 14 weeks. Selected leaves were subjected for phenotypical analysis. (A) Representative plants of the indicated genotypes showing different degrees of leaf senescence (yellowing). (B-C) Representative micrographs showing a leaf section of the 10 th leaf of Col-0, eps and eps3m , and the 14 th leaf of 3m/ C after 3,3’-diaminobenzidine staining for in situ detection of H 2 O 2 (brownish precipitates in B) or trypan blue staining for cell death (dark blue in C). Bars=50 μm. (D) Representative micrographs showing Gc UCSC1-infected 11 th leaves of Col-0, eps and eps3m , and the 15 th leaves of 3m/ Col-0 plants at 3 dpi after trypan blue staining. Red arrows indicate germinated sporelings that failed to develop further; purple arrows indicate incidental trichome-supported fungal growth. Bars=100 μm. To further evaluate this inference, we tested eps and eps3m seedlings with a virulent oomycete pathogen, Hyaloperonospora arabidopsidis Noco2. Plants of both genotypes were similarly susceptible to this pathogen ( Fig. 3C ). This further suggest that the failure of PM fungi in eps3m plants is not due to activation of defenses. We then created eps3m transgenic plants expressing MLO2-GFP from the MLO2 promoter and found that such transgenic plants largely restored susceptibility to Gc UCSC1 ( Fig. 3D ), reinforcing the finding that MLO2 is the major contributor to PM susceptibility among the three clade V MLO genes ( Consonni et al., 2006 ). Failure of PM infection on eps3m is not associated with defense activation To further ascertain whether the near-complete resistance of eps3m to Gc UCSC1 can be uncoupled from host defense, we first grew plants of Col-0, 3m/C , eps , and eps3m under short-day condition for 14 weeks when old rosette leaves of Col-0 wild-type plants started to show weak leaf yellowing (senescence) ( Fig. 4A ). At this time, plants of 3m/C showed massive leaf senescence and reduce stature as anticipated, whereas plants of eps and eps3m showed no sign of leaf senescence ( Fig. 4A ). Leaf senescence initiation is known to be associated with reactive oxygen species (ROS) production and accumulation in mesophyll cells that eventually leads to collapse of mesophyll and epidermal cells, resulting in leaf yellowing, a typical phenotype of senescence ( Jing et al., 2008 ; Mayta et al., 2019 ). To see if there is ROS accumulation in leaves of eps3m plants, we subjected the 10 th leaf from plants of Col-0, eps and eps3m , and the14 th leaf of 3m/C to 3,3’-diaminobenzidine (DAB) staining for visualization of in situ H 2 O 2 accumulation. While the 10 th leaf of the plants of Col-0, eps and eps3m showed no or little (in the case of Col-0) visible yellowing, the 10 th to the 13 th leaves of 3m/C plants exhibited obvious yellowing and their 14 th leave showed little yellowing. Subsequent microscopy revealed frequent H 2 O 2 -positve individual and clustered mesophyll cells in the 14 th leaves of all six 3m/C plants examined ( Fig. 4B ), indicative of the onset of senescence in these leaves. In contrast, such H 2 O 2 -positive cells were completely absent from the 10 th leaves of eps and eps3m plants, and only rarely (∼5% leaf areas) observed in the 10 th leaves of Col-0 plants ( Fig. 4B ), indicating (largely) absence of senescence. We then used trypan blue staining to visualize dead or dying cells in the 10 th or 14 th leaves of these four genotypes and found very similar patterns as observed for H 2 O 2 accumulation: while cell death was not detected from the 10 th leaves of eps and eps3m plants, and only rarely seen in the 10 th leaves of Col-0 plants, collapse of individual and clustered mesophyll cells, and occasionally more than a dozen of both mesophyll and epidermal cells, was frequently found in the 14 th leaves of 3m/C plants ( Fig. 4C ). To examine if PM inoculation can trigger cell death in 3m/C , and whether the cell death contributes to the termination of fungal development in 3m/C , we inoculated the detached 11 th or 15 th leaves of these 14-week-old plants with Gc UCSC1 and examined the host-fungal interaction by trypan blue staining at 3 dpi. As shown in Fig. 4D , normal fungal development occurred without cell death in the 11 th leaves of the susceptible Col-0 and eps plants, whereas the sporelings were completely arrested shortly after germination on pavement cells of both the 15 th leaves of 3m/C and 11 th leaves of eps3m (indicated by red arrows in Fig. 4D ). Notably, while no cell death was observed in eps3m , the cell death in 3m/C was not associated with the early termination of fungal development. As observed before, sporadic hyphal development could be supported by trichome cells in both 3m/C and eps3m (indicated by purple arrows in Fig. 4D ). The above observations suggest that (i) the failure of Gc UCSC1 in colonizing host plants due to the loss of MLO2, MLO6 and MLO12 is independent of ROS production and cell death associated with leaf senescence and (ii) early senescence-associated ROS production and cell death due to the impairment of the three MLO genes is EDS1 , PAD4 and SID2 -dependent. The latter agrees with the observation that mlo2 -conditioned early leaf senescence is PAD4 - and SA-dependent ( Consonni et al., 2006 ). Next, we analyzed the expression of four marker genes in mature leaves of seven-week-old short-day-grown plants of Col-0, 3m/C , eps , and eps3m prior to and 6, 12 or 48 hrs post-inoculation (hpi) with Gc UCSC1. Before inoculation, none of the plants exhibited signs of early senescence. Four marker genes, FRK1 , PR1 , PDF1.2 and SAG101 were chosen: FRK1 (Flg22-Induced Receptor-Like Kinase 1) is a marker of PTI activation ( Asai et al., 2002 ), PR1 induction reports the activation of the SA-dependent defense responses during ETI (and PTI) and systemic acquired resistance (SAR) ( Jing et al., 2008 ; Tsuda et al., 2013 ), PDF1.2 is a marker for the activation of the jasmonic acid (JA) and ethylene (ET) pathways ( Penninckx et al., 1998 ), while SAG101 is a senescence-associated marker gene ( He and Gan, 2002 ). As shown in Supplementary Fig. 5A and B, prior to inoculation with Gc UCSC1, expression of FRK1 in 3m/C was higher, though not statistically significant, than that in Col-0, suggesting weak constitutive activation of PTI in 3m/C plants. At 6 hpi, FRK1 was induced to higher levels in all the four genotypes. At 12 hpi, while the expression of FRK1 reached significantly higher in 3m/C , it subsided almost to basal levels in Col-0, eps and eps3m . The patterns of expression of PR1 in the four genotypes are similar to those of FRK1 , except that PR1 expression in all the four genotypes was low at 6 dpi across the board. These observations suggest that functional impairment of the three MLO genes led to pronounced PTI and likely ETI, both of which are mainly EDS1/PAD4/SID2-dependent, as eps and eps3m had no or little induction of FRK1 and PR1 expression before or after inoculation. Expression of PDF2.1 was barely detectable in plants of all the four genotypes prior to inoculation and induced to higher levels to varied degrees in the four genotypes. However, there was no significant differences among the four genotypes at any time points ( Supplementary Fig. S5C ). Expression of SAG101 remained stable and was even slightly reduced after PM inoculation, and there were no significant differences among the four genotypes at any time points ( Supplementary Fig. S5D ), which is consistent with the lack of any visible leaf senescence in these plants during the period of this experiment. Taken together, the data shown above reinforce the notion that MLO2 , MLO6 , and MLO12 may negatively regulate EDS1 / PAD4 - and SA-associated defense. Importantly, the similar expression of the four marker genes in eps and eps3m ( Supplementary Fig. S5 ) suggests that mlo -mediated ‘resistance’ to PM fungi is unlikely due to the activation of either PTI or ETI. MLO2 dosage positively correlates with host susceptibility to Gc UCSC1 We noticed that the T1 plants of eps3m mutants expressing MLO2-GFP from the native MLO2 promoter showed varied degrees of susceptibility to Gc UCSC1, ranging from near the susceptibility level of eps plants to complete lack of infection ( Fig. 5A,B ). This implies that MLO2 may facilitate PM pathogenesis in a dosage-dependent manner, which could be inferred from the reported differential levels of susceptibility of the transgenic mlo lines overexpressing an orthologous wheat or rice MLO gene ( Piffanelli et al., 2004 ; Elliott et al., 2005 ; Ge et al., 2020 ). To definitively determine if the degree of susceptibility in the T1 lines positively correlates with the levels of MLO2-GFP, we performed a western blot to assess the MLO2-GFP levels using an anti-GFP antibody in Gc UCSC1-inoculated plants of the four independent eps3m-MLO2-GFP transgenic lines shown in Fig. 5A,B . The full length MLO2-GFP (with an expected size of 93 kDa) was not detectable; however, two smaller bands (∼35 kDa) with ascending intensity were detected in the four lines with increased levels of susceptibility ( Fig. 5B-C ). Given that GFP is ∼27 kDa, the cleavage products are ∼8 kDa. Because the levels of these two small bands most likely reflect those of the full length MLO2 protein, it is most likely that the MLO2 dosage in those transgenic plants positively correlates with their levels of susceptibility to Gc UCSC1. In this context, it is interesting to note that a C-terminal cleavage product was also detected in E coli- expressed GST-tagged C-terminus of MLO2 and the cleavage product was diminished when two conserved residues of the C-terminus were mutated (von Bogartz et al., 2023). Whether such cleavage has biological relevance to MLO2’s functionality remains to be determined. Download figure Open in new tab Figure 5. MLO2-GFP expression levels positively correlate with susceptibility to Gc UCSC1 (A) Representative leaves of four selected epsm3 lines transgenic for MLO2p::MLO2-GFP showing different levels of susceptibility to Gc UCSC1. Photos were taken at 11 dpi. DR, disease reaction score judged visually based on leaf coverage of fungal mass. (B) Quantification of total number of spores per mg of infected leaf tissues of the four lines. Different letters indicate statistically significant differences ( P <0.05) between the three lines, as determined by multiple comparisons using one-way ANOVA, followed by Tukey’s HSD test. This experiment was repeated once with similar results. (C) Western blot using anti-GFP antibody to measure MLO2-GFP protein levels in the four lines. The eps3m parental line served as negative control. Numbers above the bands indicate intensity of the two bands against the blank background measured with ImageJ. ER/Golgi-localized MLO2 is targeted to peri-penetration peg membranous space Barley HvMLO-GFP and Arabidopsis MLO2-GFP have been shown to accumulate at the fungal penetration site ( Bhat et al., 2005 ; Qin et al., 2020 ). Because the term “penetration site” is rather vague, we sought to determine MLO2’s subcellular localization with higher spatiotemporal resolution to better understand the cellular functions of MLO2 in facilitating PM pathogenesis. To this end, we first inoculated plants of the eps3m - MLO2-GFP transgenic line #4 with Gc UCSC1 for localization analysis. A close microscopic examination of infected leaves stained with propidium iodide at 2 dpi revealed accumulation of MLO2-GFP in a collar-like structure around the penetration peg underneath the appressorium, with MLO2-GFP puncta distributing around the penetration site ( Fig. 6A, B ). To better examine MLO2-GFP’s dynamic spatiotemporal localization during appressorium-haustorium differentiation, we introduced the pRPW8.2::RPW8.2-RFP construct into the eps3m - MLO2-GFP background. RPW8.2 is specifically targeted to the extra-haustorial membrane (EHM) encasing the haustorium that emanates from the tip of the penetration peg, hence RPW8.2 can serve as a reporter of the spatiotemporal biogenesis of the EHM and the haustorium ( Wang et al., 2009 ). A time course analysis showed that PM spores germinated at ∼6 hpi and MLO2-GFP first exhibited detectable focal accumulation at 7.5 hpi ( Fig. 6C,D ) whereas RPW8.2-RFP was first detectable at the EHM around 16 hpi ( Fig. 6E ) ( Wang et al., 2009 ). Importantly, MLO2-GFP was observed to accumulate in a compartment (∼1-2 μm thick and 1-3 μm long) surrounding the penetration peg. We tentatively named this compartment the p eri- p enetration-peg m embranous space (PPM). Notably, MLO2-GFP was never found in the EHM which initiates from the haustorial neck connecting to the penetration peg ( Fig. 6F-H ; Supplementary Fig. S6 ). These observations imply that MLO2 is specifically recruited to the p eri- p enetration-peg m embranous space (PPM) ( Fig. 6I,J ) where it may play an important role in sealing and stabilizing the convoluted membrane junction, thereby accommodating haustorium differentiation (See Discussion). Download figure Open in new tab Figure 6. MLO2-GFP is localized to the peri-penetration peg membranous space (PPM). Plants of eps3m expressing MLO2-GFP (A,B) or plants of eps3m expressing both MLO2-GFP and RPW8.2-RFP (C-H) were used to determine MLO2-GFP’s localization by confocal imaging. All images are Z-stack projections of 5 to 15 optical sections. Bar=10 μm. (A,B) Representative images showing localization of MLO2-GFP to the plasma membrane of a leaf epidermal cell penetrated by Gc UCSC1. Image in (A) is a top-down view of a fungal penetration site, whereas image in (B) is a side view of the same site. Insets are closeup view of a single optical section. The fungal structure is stained red by propidium iodide. White arrows indicate MLO2-GFP puncta which may be MVBs or endosomes. White asterisk indicates penetration peg, while dark asterisk indicates haustorial neck. CW, cell wall. (C) A representative image showing punctum distribution of MLO2-GFP at 6 hpi. (D) A representative image showing focal accumulation of MLO2-GFP at 7.5 hpi. (E) A representative image showing localization of MLO2-GFP and RPW8.2-RFP at 16 hpi. White arrowhead indicates the plasma membrane, while magenta arrowhead indicates the extra-haustorial membrane (EHM). (F-H) Images showing MLO2-GFP accumulates around the penetration peg, next to the haustorial neck (also see Supplementary Fig. S4 ). (I) A cartoon depicting the focal accumulation of MLO2 from Golgi bodies to the penetration site. Pp, penetration peg; Hn, haustorial neck; MVB MLO2 , multivesicular bodies (MVBs) containing MLO2; EHM, extra-haustorial membrane. (J) A zoom-in cross section of (I) illustrating the membrane junction where MLO2 focally accumulates. We propose that exosomes derived from MVBs MLO2 are secreted into the space between the penetration peg and the host cell wall and plasma membrane (PM) to seal this membrane junction. We designate this junction filled with MLO2-containing exosomes or paramural bodies as the p eri- p enetration peg m embranous space (PPM). PPM may also include the perturbed PM and the haustorial neckband (Nb), which is believed to be a diffusion barrier between the apoplast and the extra-haustorial membrane matrix. Ectopic expression of MLO7 but not MLO1 partially restores susceptibility of eps3m to Gc UCSC1 Several MLO family members in Arabidopsis exhibit distinct expression patterns and serve distinct biological functions ( Chen et al., 2006 ; Davis et al., 2017 ; Jones and Kessler, 2017 ; Li and Xiao, 2025 ). Given that MLO1, MLO2 and several other MLOs assayed possess calcium channels activity when ectopically expressed in mammalian cells ( Gao et al., 2022 ; Gao et al., 2023 ), we wondered if MLOs belonging to other clades and expressing in other organs/trissues share the same molecular functions as MLO2 in facilitating PM pathogenesis when ectopically expressed in leaf epidermal cells. Since the eps triple mutant is super-susceptible to Gc UCSC1, whlie eps3m is essentially “immmue” to Gc UCSC1, we reasoned that any functional complementation of the loss of MLO2/6/12 in eps3m should be readily manifested by fungal growth visible to the nake eye. To test this, we made eps3m transgenic plants expressing barley MLO1 ( HvMLO1 ) from the 35S promoter and found that nine of 20 T1 transgenic plants supported varied degree of susceptibility to Gc UCSC1 ( Supplementary Fig. S7A ), indicating that HvMLO1 can largely perform the same molecular function as MLO2 in Arabidopsis, despite that it belongs to a different clade (clade IV) in the MLO family tree ( Kusch et al., 2016 ). We then made transgenic eps3m lines expressing MLO6-GFP from the MLO6 promoter. Five of 14 Gc UCSC1-infected T1 plants transgenic for MLO6-GFP showed visible but limited fungal growth ( Fig. 7A ) with ∼10% of spore-production compared to those expressing MLO2-GFP from the MLO2 promoter ( Supplementary Fig. S7B ,C). Confocal imaging showed that MLO6-GFP also exhibited similar focal accumulation at the fungal penetration site in the five transgenic lines ( Fig. 7B ). These results support the notion that MLO2 play a major role while MLO 6 and MLO12 play a minor role in permitting PM pathogenesis ( Consonni et al., 2006 ) and that protein accumulation at PPM may be a common feature of MLOs that function as susceptibility factors. The above results also demonstrate that PM infection in epsm3 transformants can be used as a sensitive reporter to evaluate whether any candidate wild-type or mutant MLO genes can perform the same molecular function as MLO2 by ectopically expressing it in leaf epidermal cells from the MLO2 promoter. Download figure Open in new tab Figure 7. Functional complementation tests for other MLO family members in eps3m . Plants of eps3m lines transgenic for the indicated MLOx-GFP DNA constructs were inoculated with Gc UCSC1. Infection phenotypes were visually and microscopically examined. Plant photos were taken at 10-11 dpi. Infected leaves were subjected to confocal imaging at 3 dpi for assessing the localization of each MLOx-GFP fusion protein. Images shown are Z-stack projection of 3-5 optical sections. Fungal structures were stained red with propidium iodide. Arrows indicate penetration sties. Bar=50μm. A similar strategy was employed to show that expression of MLO2 in synergid cells from the MLO7 ( NTA ) promoter significantly restored the fertility of the mlo7 mutant ( Jones et al., 2017 ), suggesting that MLO2 and MLO7 share similar molecular function. To provide concrete evidence for the converse scenario, we generated eps3m plants expressing MLO7-GFP from the MLO2 promoter and found that five of 18 T1 plants were moderately susceptible and six T1 plants were weakly susceptible to Gc UCSC1 (Fig., 7C; Supplementary Fig., S7D). Not surprisingly, like MLO2-GFP, MLO7-GFP also exhibited focal accumulation at the PPM ( Fig. 7D ). This indicates that MLO7 can partially complement the loss of MLO2/6/12 if ectopically expressed in leaf epidermal cells. To further expand such functional assays to other MLO clades, MLO4 and MLO11 (belonging to clade I), MLO1 (belonging to clade II), MLO5 and MLO10 (belonging to clade III), and MLO3 (belonging to clade VI) were cloned into the same binary vector that contains the MLO2 promoter for translational fusion with GFP ( Supplementary Fig. S8 ). At least eight eps3m T1 transgenic plants were produced for each construct. All T1 plants were inoculated with Gc UCSC1 to determine if (i) any plants can support fungal growth and sporulation and (ii) whether the fusion proteins are detectable by confocal microscopy and if so, where they are localized. Intriguingly, none of the T1 plants expressing any of the six tested MLO genes ( MLO1 , MLO3 , MLO4 , MLO5 , MLO10 and MLO11 ) supported visible growth of Gc UCSC1 ( Fig. 7E, G, I, K ) and no GFP signal was reliably detected in the T1 plants transgenic for MLO3-GFP , MLO4-GFP and MLO5-GFP ( not shown ). Among the 60 eps3m T1 plants expressing MLO1-GFP , GFP signal was readily detected in the plasma membrane (labeled by the lipophilic dye FM4-64) of leaf epidermal cells ( Supplementary Fig. S9A ), which is consistent with the MLO1-GFP localization pattern observed in epidermal cells of Nicotiana benthamiana leaves after agrobacterium-mediated transient expression ( Jones and Kessler, 2017 ). Notably, sporelings were arrested probably due to failure in host entry in eps3m plants expressing MLO1-GFP and there was no detectable change of MLO1-GFP in response to attempted fungal penetration ( Fig. 7F ). Similarly, GFP signal was detected in the plasma membrane of the leaf epidermal cells in the eight eps3m T1 plants transgenic for MLO11-GFP ( Supplementary Fig. S9B ) and the sporelings failed to develop further ( Fig. 7H ). Interestingly, although no fungal mass was visible to the naked eye in any of the 15 eps3m T1 plants transgenic for MLO10-GFP ( Fig. 7I and K ), a few small fungal colonies with very limited hyphal growth without conidiophore formation were detectable in T2 progenies of two independent T1 lines ( Fig. 7L ). MLO10-GFP was detected in puncta and likely at the plasma membrane, but there was no obvious focal accumulation even in the case of successful penetration reported by limited mycelial growth ( Fig. 7L ). These observations suggest that in term of subcellular localization, MLO10 is similar to MLO2 but distinct from MLO1 and MLO11. Given that MLO10 was able to restore fertility of mlo7 when expressed in synergid cells ( Jones et al., 2017 ), the inability of MLO10-GFP to significantly complement PM susceptibility in the eps3m background was unexpected, especially given its close homology to MLO7. To validate the three MLO-GFP fusion constructs, i.e., MLO3-GFP , MLO4-GFP and MLO5-GFP, whose expression was not detectable in their respective Arabidopsis transgenic plants, and assess their subcellular localization, we transiently co-expressed them with HDEL-mCherry (an ER marker) and Man1 (1-49)-mCherry (a Golgi marker) ( Nelson et al., 2007 ) in N. benthamiana leaves via agroinfiltration. Confocal imaging at three days post-infiltration revealed that all three fusion proteins were detectable and exhibited similar patterns of partial co-localization with HDEL-mCherry and/or Man1-mCherry ( Supplementary Fig. S10 – 12 ). These observations suggest that MLO3-GFP, MLO4-GFP, and MLO5-GFP are also likely localized to the ER and/or Golgi compartments in Arabidopsis ; however, their low expression and/or rapid turnover may prevent reliable detection in stable eps3m transgenic plants. Domain-swapping between MLO1 and MLO2 reveals the C-terminal CaM-binding domain as a key determinant of subcellular localization MLO7 (i.e., NTA) localizes to Golgi bodies in synergid cells and redistributes to the plasma membrane at the filiform apparatus during pollen tube reception ( Jones et al., 2017 ; Ju et al., 2021 ). A chimeric MLO protein that contains the N-terminal 7TM portion of NTA and the cytoplasmic CaMBD-containing C-terminus of MLO1 (designated faNTA) exhibits constitutive localization at the filiform apparatus and is able to restore the fertility of nta ( Ju et al., 2021 ). This finding suggests that the C-termini of MLO1 and MLO7 determines their respective localization. To determine whether the CaMBD-containing C-termini of MLO1 and MLO2 dictate their respective subcellular localization in leaf epidermal cells, we constructed two chimeric genes by swapping the fragments encoding their C-terminal domains. The first resulting chimeric protein consists of MLO1’s N-terminal 7TM (1-438 amino acids) and MLO2’s C-terminus (442-574 amino acids) (designated MLO1n-2c), while the second comprises MLO2’s N-terminal 7TM (1-442 amino acids) with MLO1’s C-terminus (439-526) (designated MLO2n-1c) ( Fig. 8A ). These chimeric genes, MLO1n-2c and MLO2n-1c , were translationally fused with GFP at their C-termini, and the resulting fusion constructs were stably expressed in eps3m from the MLO2 promoter. More than 20 eps3m T1 plants transgenic for either of the two chimeric fusion genes were generated and inoculated with Gc UCSC1. None of the 23 T1 plants transgenic for MLO1n-2c-GFP supported any visible fungal growth and propidium iodide staining showed that sporelings were arrested shortly after germination ( Fig. 8B ), and MLO1n-2c-GFP exhibited punctum distribution ( Fig. 8C ). To test if successful penetration followed by haustorial differentiation can induce MLO1n-2c-GFP’s focal accumulation, we introduced the same MLO2p::MLO1n-2c-GFP construct into the eps background to allow growth of Gc UCSC1. The T1 transgenic plants were as susceptible as eps ( Fig. 8D ), suggesting that MLO1n-2c expression has no dominant negative effect. Interestingly, MLO1n-2c-GFP was found to accumulate at the PPM in the eps transgenic plants ( Fig. 8E ). By contrast, seven out of 21 e psm3 T1 plants transgenic for MLO2n-1c-GFP supported fungal hyphal growth as revealed by propidium iodide staining at 3 dpi ( Fig. 8G ), and three of them also supported weak Gc UCSC1 infection visible to the naked eye at 13 dpi ( Fig. 8F ). Interestingly, similar to MLO1-GFP ( Fig. 8F ), MLO2n-1c-GFP exhibited plasma membrane localization in leaf epidermal cells, and there was no obvious focal accumulation around the fungal penetration site ( Fig. 8G ). Collectively, the above results indicate that the C-terminus of MLO1 confers plasma membrane localization whereas the C-terminus of MLO2 endows punctum distribution and fungal penetration-induced focal accumulation at the PPM. The results also suggest that (i) the N-terminal 7TM portion of MLO2, but not that of MLO1, specifies the function of MLO2 in accommodating entry of the PM pathogen and (ii) the full function of MLO2 requires its enrichment at the PPM, which is regulated by its CaMBD-containing C-terminus. Download figure Open in new tab Figure 8. Domain-swapping analysis between MLO1 and MLO2 reveals two functional arms of MLO2. (A) Schematic illustration of the experimental design. (B,D,F) Infection phenotypes of representative plants expressing MLO1n-2c-GFP or MLO2n-1c-GFP from the MLO2 promoter in either the eps3m background (B,F) or the eps background (D). Photos were taken at 12 dpi with Gc UCSC1. Arrows in (F) point to infected leaf tips with visible fungal mass. (C,E,G) Representative confocal images showing localization of the indicated chimeric fusion proteins in leaf epidermal cells at 3 dpi. Images are Z-stack projection of 3-5 optical sections. Fungal structures were stained red with propidium iodide. Inset in E is a closeup view of a single penetration site where MLO1n-2c accumulates. Bar=50μm. Loss of FERONIA does not affect MLO2’s localization and role in facilitating PM pathogenesis FERONIA (FER), a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein subfamily ( Escobar-Restrepo et al., 2007 ; Guo et al., 2009 ), has been shown to be important for the proper localization of MLO7 in synergid cells ( Ju et al., 2021 ). Additionally, fer mutants exhibit reduced susceptibility to PM disease ( Kessler et al., 2010 ). Given the conserved molecular functions shared by MLO2 and MLO7 ( Fig. 7C ; Jones et al., 2017 ), we asked whether FER is also required for the focal localization of MLO2 at the PPM in leaf epidermal cells. To test this, we first performed CRIPSR-targeted mutagenesis in the eps background to determine if FER is required for MLO2 function and if so, eps-fer mutants should exhibit reduced susceptibility to Gc UCSC1 as seen in the five mlo2 cipi mutants ( Fig. 1 ). Loss-of-function fer mutants displays a more compact rosette phenotype readily distinguishable from wild-type plants ( Deslauriers and Larsen, 2010 ). We obtained seven presumable eps - fer mutants with a compact rosette and found that all of them showed similar levels of susceptibility to Gc UCSC1 as eps plants ( Fig. 9A ). Sequencing three of the seven mutants revealed disruptive indel mutations close to the sgRNA target sites in the second exon of FER ( Supplementary Fig. S13A ). This result indicates that, unlike MLO2 , FER is dispensable for PM pathogenesis in eps and implies that the reported enhanced resistance in fer single mutants ( Kessler et al., 2010 ) may be due to activation of EDS1/PAD4/SID-dependent immunity. Next, we introduced the same CRISPR/Cas9 construct into eps3m plants transgenic for MLO2-GFP and RPW8.2-RFP to determine if focal accumulation of MLO2-GFP is affected in the absence of FER. Among 27 T1 transgenic plants obtained, 18 displayed the compact rosette phenotype. Among the 18 plants, eight were susceptible to Gc UCSC1, the remaining10 plants did not support any growth of Gc UCSC1 visible to the naked eye ( Fig. 9B ). Targeted sequencing of FER in three of the eight susceptible plants with a compact rosette identified disruptive indels in FER as expected ( Supplementary Fig. S13B ). Confocal imaging showed that MLO2-GFP signal was not detectable in the 10 T1 plants with a compact rosette but lacking infection, suggesting that the MLO2-GFP transgene is silenced, which was frequently observed in transgenic plants generated in the eps3m - MLO2-GFP;RPW8.2-RFP background (data not shown). Importantly, MLO2-GFP, as well as RPW8.2-RFP in the eight T1 plants susceptible to the fungus exhibited normal localization ( Fig. 9C,D ), indicating that loss of FER does not affect MLO2’s localization to the PPM and its role in accommodating PM pathogenesis in leaf epidermal cells. To assess if there is functional redundancy among FER and its family members, we performed multiplexed CRISPR to target eight CrRLK1L family members including FER that are known to be involved in immunity and/or expressed in leaves based on the search results from the RNA-seq database which include >20,000 RNA-seq libraries ( https://plantrnadb.com/athrdb/ ). Among 25 T1 transgenic lines, 11 displayed the compact rosette phenotype, among which five were susceptible ( Fig. 9E ). We selected one of the susceptible lines for sequencing of all the eight targeted CrRLK1L genes. The results showed that this line contains disruptive mutations in FER and five additional CrRLK1Ls ( Fig. 9F ; Supplementary Fig. S14 ), yet MLO2-GFP’s localization and role in accommodating PM entry remain unaffected (not shown), indicating that those six CrRLK1Ls are dispensable for MLO2’s localization and function. Download figure Open in new tab Figure 9. FERONIA and its five family members are dispensable for MLO2’s focal accumulation and MLO2-mediated susceptibility. CRISPR-targeted mutagenesis was used to knock out Feronia ( FER ) alone, or FER and other five members of the CrRLK1L gene family in eps or eps3m expressing MLO2-GFP and RPW8.2-RFP. Independent T1 plants were inoculated with Gc UCSC1 and plant photos were taken at 10-12 dpi. Mutations in FER and other family member were identified by targeted sequencing (see Supplementary Fig. S10 and S11 ). (A) Infection phenotypes of representative eps plants and three T1 transgenic lines of eps3 expressing the CRISPR construct targeting FER (a1-a3). Sanger sequencing results are shown in Supplementary Fig. S10A ) (B) Infection phenotypes of T1 plants of eps3m/pMLO2-MLO2-GFP/pRPW8.2-RPW8.2-RFP transgenic for the CRISPR construct targeting FER . Three susceptible fer- like mutant plants (b1-b3) were subjected to sequencing analysis of FER (see Supplementary Fig. S10B ). (C,D) Representative confocal images showing typical focal accumulation of MLO2-GFP (C) and EHM-localization of RPW8.2-RFP (D). Images are Z-stack projection of 3-5 optical sections. Fungal structures were stained red with propidium iodide. Bar=20μm. (E) Infection phenotypes of T1 plants of eps3m/pMLO2-MLO2-GFP/pRPW8.2-RPW8.2-RFP transgenic for the CRISPR construct targeting eight CrRLK1L family genes. Three susceptible fer- like mutant plants (e1-e3) were subjected to sequencing analysis to reveal the indel mutations. (F) A chart showing the mutations in the eight CrRLK1L genes targeted by CRISPR mutagenesis in line e2 shown in (E). The Sanger sequencing chromatograms are shown in Supplementary Fig. S11 . Loss of Phosphatidylinositol 4-phosphate 5-kinase 1 (PIP5K1) and PIP5K2 does not significantly affect the function and localization of MLO2 Qin et al. (2020) reported that the loss of PIP5K1 and PIP5K2 significantly impaired MLO2-GFP’s recruitment to the fungal penetration site, resulting in greatly enhanced resistance to an adapted PM fungus ( Qin et al., 2020 ). Based on these findings, they proposed that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], the product of PIP5K1 and PIP5K2, may be required for MLO2’s focal accumulation, thereby acting as a host susceptibility factor of powdery mildew ( Qin et al., 2020 ). To determine if MLO2’s focal accumulation and function requires PIP5K1 and PIP5K2, we knocked out PIP5K1 and PIP5K2 by CRISPR in the eps3m line transgenic for pMLO2 :: MLO2-GFP and pRPW8.2::RPW8.2-RFP and found that the pip5k1-pip5k2 double mutant plants with greatly reduced stature were also very susceptible to Gc UCSC1 ( Fig. 10A ). Close examination revealed that the first 2-3 true leaves of the mutants were dark purple and often showed reduced infection ( Fig. 10B ). Sequencing of 10 of 17 independent mutant T1 lines with greatly reduced stature ( Supplementary Fig. S15 ) confirmed the presence of homozygous or biallelic mutations in PIP5K1 and PIP5K2 , with the chromatograms of the sequenced mutation sites from two lines (C6 and L16) shown in Fig. 10C . Confocal imaging of infected leaves of the two mutant lines confirmed typical localization of MLO2-GFP at the PPM and normal of RPW8.2-RFP at the EHM ( Figure 10D ). Collectively, these results demonstrate that PIP5K1 and PIP5K2, and the PI(4,5)P 2 pool derived from these two enzymes, are dispensable for MLO2’s localization and function. Download figure Open in new tab Figure 10. Loss of PIP5K1/K2 does not affect MLO2’s focal localization and MLO2-mediated susceptibility. CRISPR-targeted mutagenesis was used to knock out PIP5K1 and PIP5K2 in eps3m expressing MLO2-GFP and RPW8.2-RFP. Independent T1 plants were inoculated with Gc UCSC1 and plant photos were taken at 10 dpi. Mutations in these two genes in 10 transgenic plants with small stature were identified by targeted sequencing, and two of which were shown in (C). (A) Two representative transgenic plants with greatly reduced stature were similarly susceptible as the bigger plants of the parental line. (B) A closeup view of a putative pip5k1/2 mutant plant showing reduced infection of the first 2-3 true leaves (arrows) with anthocyanin accumulation. (C) Four chromatographs showing disruptive indels in PIP5K1 and PIP5K2 in two selected mutant plants. Long arrowed lines indicate small guide RNAs; short arrows point to the inserted nucleotides. (D) A representative confocal image showing normal localization of MLO2-GFP and of RPW8.2-RFP. Bar=20μm. Discussion MLOs have long been considered to be host susceptibility or compatibility factors of powdery mildew ( Panstruga, 2003 ). Yet, to date, there is no definitive genetic evidence to exclude ectopic activation of defense as the molecular basis of mlo -mediated “resistance” despite that no specific defense pathways or components have vigorously been shown to contribute to the “resistance” of the mlo2/mlo6/mlo12 triple mutants ( Kuhn et al., 2017 ). In this study, through a tailored forward genetic screen that aims to identify mutants that display compromised-immunity-yet-poor infection ( cipi ) phenotypes, we discovered disruptive mutations in MLO2 to be responsible for the cipi phenotype of five best cipi mutants. CRISPR-targeted mutagenesis of MLO6 and MLO12 in cipi3 and subsequent analyses further demonstrate that mlo2/6/12 -conditioned suppression of infection can be uncoupled from ectopic activation of plant defenses, providing definitive genetic and molecular evidence to a long-awaited conclusion that MLO proteins are bona fide host susceptibility factors that are essential for PM pathogenesis. Uncoupling of defense activation from mlo -conditioned failure of PM pathogenesis The concept of host susceptibility factors essential for pathogenesis has long been established in the case of viruses ( Strauss and Strauss, 1999 ). Both RNA and DNA viruses depend on host machinery for their replication, hence mutagenesis of relevant host translation initiation / elongation factors has long been considered effective strategies to engineer viral resistance, particularly in plants ( Sanfacon, 2015 ). A good example of this strategy is provided by a recent report on engineering resistance to maize lethal necrosis caused by maize chlorotic mottle virus along with a potyvirus ( Wen et al., 2024 ). Many “susceptibility” genes have been identified in various plant species that their loss hinders pathogenesis of bacterial, fungal, oomycete pathogens, creating opportunities for engineering plant resistance through targeted mutagenesis ( Garcia-Ruiz et al., 2021 ; Koseoglou et al., 2022 ). For example, disruption of DMR6 , which encodes a 2-oxoglutarate (2OG)-Fe(II) oxygenase of previously unknown function, renders Arabidopsis resistant to Hyaloperonospora parasitica , an obligate biotrophic oomycete ( van Damme et al., 2008 ). Subsequent studies revealed that loss of DMR6 homologs leads to salicylic acid (SA)-dependent immune activation and broad-spectrum disease resistance across multiple plant species ( Kieu et al., 2021 ; Thomazella et al., 2021 ; Tripathi et al., 2021 ; Giacomelli et al., 2023 ; Zhang et al., 2025 ). Thus, dmr6 -mediated resistance is mostly due to activation of plant immunity. Another group of well-characterized susceptibility genes encode SWEET sugar transporters, which are exploited by Xanthomonas oryzae pv. oryzae —the causal agent of rice bacterial blight—through TAL effectors to increase host sugar availability. Natural recessive mutations or CRISPR-enabled editing of SWEET genes or their promoters can effectively block sugar exploitation by the bacterial pathogen, thereby conferring resistance to bacterial blight in rice ( Chu et al., 2006 ; Yang et al., 2006 ; Oliva et al., 2019 ; Schepler-Luu et al., 2023 ). The great potential of controlling devasting diseases through identification and targeted interference of susceptibility genes of the causative pathogens was further demonstrated by an elegant study focusing on citrus greening ( Zhao et al., 2025 ). Zhao and colleagues discovered a key susceptibility gene, encoding an E3 ubiquitin ligase, PUB21, and a transcription factor MY2 regulating JA-dependent defense response as the substrate of PUB21. Based this pathogenicity mechanism, the authors searched and identified a 14–amino acid peptide, APP3-14, that can bind and inhibit PUB21 activity, thereby stabilizing MYC2 and ensuring induction of JA-dependent resistance against the causal bacterial pathogen of citrus greening ( Zhao et al., 2025 ). This highlights the importance of understanding the role of host susceptibility factors in pathogenesis. However, although recessive mlo mutation-mediated “resistance” to PM fungi was characterized in 1997 ( Buschges et al., 1997 ) and has been widely exploited in various crop species ( Kuhn et al., 2017 ; Kusch and Panstruga, 2017 ; Li et al., 2022 ), its underlying mechanisms remain largely elusive. It is notable that mlo -mediated resistance is restricted to clade IV and V almost in all cases [summarized in table 1 in ( Li and Xiao, 2025 )] even though MLO7, a clade III MLO expressed in synergid cells, can partially complement the loss of MLO2, MO6 and MLO12 in leaf cells ( Fig. 7C ). Depending on the pathosystems used for study, clade IV and V mlo mutants have been shown to be either more susceptible to hemi-biotrophic or necrotrophic pathogens (see a recent review by ( Li and Xiao, 2025 )). For example, mlo2/mlo6/mlo12 triple mutants showed increased susceptibility to hemi-biotrophic bacterium Pseudomonas syringae ( Acevedo-Garcia et al., 2017 ), necrotrophic fungi Alternaria alternata and A. brasscisicola , and hemi-biotrophic oomycete Phytophthora infestans ( Consonni et al., 2006 ) and Fusarium oxysporum ( Acevedo-Garcia et al., 2017 ). However, the Arabidopsis triple mutant exhibited reduced penetration success of hemi-biotrophic Colletotrichum higginsianum , which invades host via direct cell wall penetration ( Acevedo-Garcia et al., 2017 ). Similarly, mlo mutants of barley, wheat and Medicago showed reduced root colonization by the arbuscular mycorrhizal fungus ( Jacott et al., 2020 ), conforming with the notion that clade IV/V MLOs are required for efficient host cell wall penetration by (hemi)biotrophic filamentous fungi. An exception is that the m2/6/12 triple mutants showed no altered susceptibility to obligate biotrophic oomycete Hyaloperonospora arabidopsidis and Albugo laibachii ( Acevedo-Garcia et al., 2017 )( Fig. 3C ). This may be explained by the fact that those oomycetes can enter host mesophyll layers via stomata ( Herlihy et al., 2019 ) and other MLOs expressing in mesophyll cells may serve a similar role in accommodating penetration and haustorium formation of the oomycetes in mesophyll cells. This latter theory can also explain why trichome cells of eps3m can still support limited growth of Gc UCSC1 ( Fig. 4D ). Based on the studies discussed above, along with numerous other reports [Reviewed by ( Kusch and Panstruga, 2017 ; Li and Xiao, 2025 )], a general pattern emerges regarding the biotic phenotypes resulting from disruption of clade IV/V MLOs: they are consistently associated with compromised biotrophy involving host epidermal cells. In other words, clade IV/V MLOs appear to be essential for the successful penetration of host epidermal cells by biotrophic filamentous fungi—regardless of the nature of the interaction, whether beneficial, commensal, or pathogenic. The reasoning above is strongly supported by key observations from this study, which was specifically designed to identify genes required for “biotrophy” of PM fungi in host epidermal cells. The isolation of five mlo2 allelic mutations with the strong cipi mutant phenotypes through our genetic screens suggests that MLO2 may be the single most important host protein required for PM pathogenesis ( Fig. 1 ). Consistently, the failure of PM pathogenesis in the eps3m mutant could be completely uncoupled from activation of known defenses examined ( Fig. 4 ; Supplementary Fig. S5 ). Our results also indicate that the early leaf senescence of 3m/C is due to activation of the EDS1/PAD4 and SA-dependent cellular defense programs, agreeing with earlier inferences ( Buschges et al., 1997 ; Humphry et al., 2006 ), explaining the enhanced susceptibility of mlo mutants to necrotrophic or hemi-biotrophic pathogens ( Consonni et al., 2006 ; Acevedo-Garcia et al., 2017 ). How may MLO2 work to permit PM pathogenesis? Our genetic and molecular data affirm that MLOs are essential for PM pathogenesis. A critical follow-up question one may ask is what role MLOs play in fulfilling this requirement? Given their obligate biotrophic nature, PM fungi likely co-opt an MLO-dependent host cellular process to achieve successful host penetration and haustorium differentiation. In this context, the accumulation of MLO2-GFP to the PPM, the extracellular membrane domain that matches mostly or exactly the same physical space as the papilla where callose is deposited, or the paramural body where the syntaxin PEN1 (SYP121) exosomes accumulate ( Assaad et al., 2004 ; Bhat et al., 2005 ) ( Meyer et al., 2009 ), may provide important mechanistic insights. Upon spore inoculation, the earliest focal accumulation of MLO2-GFP was detected at 7.5 hpi ( Fig. 6C, D ), which is 5-7 hours earlier than the formation of the haustorium underneath the penetration peg, which is estimated to be around 12 to 14 hpi ( Koh et al., 2005 ). Given that germinated sporelings can develop normal appressoria and penetration pegs in eps3m (pointed by an arrow in Fig. 3B ) but fail to differentiate haustoria in pavement epidermal cells ( Supplementary Fig. S4 ), we speculate that accumulation of functional V MLOs in the PPM may be a prerequisite for haustorial differentiation. More specifically, MLOs may be exocytosed into the PPM and function as scaffolding crucial for stabilizing and resealing the plasma membrane damaged by penetration, which may create a host cell environment for haustorium differentiation from the tip of the penetrate peg ( Fig. 6F-J ). In addition, a poorly characterized membranous structure, the haustorial neckband (which is indicated as a blue band in Fig. 6J ), is thought to form concomitantly with haustorial biogenesis and seal the space between the apoplast and the extra-haustorial membrane matrix ( Gil and Gay, 1977 ; Bushnell and Gay, 1978 ). Therefore, MLO2 may be also localized to this membrane and required for its formation. Higher-resolution microscopy with immunogold labeling could help confirm this. Furthermore, given MLO2’s calcium channel activity, MLO2-mediated localized calcium influx may be necessary for the resealing and stabilization of the PPM junction including neckband formation, thereby permitting haustorium differentiation inside a host cell. Functional diversification of MLOs MLO1, along with MLO5 and MLO9, is required for pollen tube integrity ( Gao et al., 2023 ), while MLO11 plays a role in root thigmomorphogenesis ( Chen et al., 2009 ). Results from our analyses through transgene expression using the MLO2 promoter demonstrate that there is clear functional diversification among MLO1 (clade II), MLO11 (clade I), MLO2, MLO6 (clade V) and MLO7 (clade III) both in terms of subcellular localization and their ability in permitting PM pathogenesis ( Fig. 7 ). While MLO2, MLO6 and MLO7 were also found in the plasma membrane of epidermal cells ( Fig. 6E and Fig. 7B,D ), they mainly exhibit punctum distribution and relocalize to the PPM in PM-infected cells to accommodate PM pathogenesis. In contrast, MLO1 and MLO11 are homogenously localized at the plasma membrane, show no obvious focal accumulation at the PPM, and cannot substitute MLO2 in permitting PM pathogenesis ( Fig. 7F,H ). Given that MLO1, MLO2, and MLO7 all exhibit calcium channel activity ( Gao et al., 2022 ; Gao et al., 2023 ), their functional diversification may stem from their regulation by different CaMs or CMLs or other proteins, rather than from their ability to conduct calcium ion across the plasma membrane. Alternatively, they do not possess calcium channel activity in epidermal cells and/or such activity is not important for their biological functions. Results from domain swapping between MLO1 and MLO2 ( Fig. 8 ) and between MLO1 and MLO7 ( Jones et al., 2017 ) suggest that there are at least two functional arms for MLO proteins: the N-terminal 7TM portion performs the cellular function of a particular MLO protein whereas the C-terminus specifies its subcellular localization. Such a functional configuration can explain the functionality of chimeric proteins MLO2n-1c in conferring partial susceptibility of eps3m to Gc UCSC1 ( Fig. 8F,G ) and NTA (MLO7)-MLO1 Cterm (also named faNTA) in restoration of fertility of mlo7 ( Ju et al., 2021 ). Notably, because MLO2n-1c does not show significant focal accumulation, its presence in the PPM is likely limited. This probably explains the weak susceptibility of eps3m lines expressing MLO2n-1c to Gc UCSC1, underscoring the importance of a “dosage” effect through MLO2’s C-terminal domain-mediated focal accumulation. This also aligns with the observed correlation between MLO2 expression levels and the susceptibility of transgenic lines ( Fig. 5 ). On the other hand, MLO1n-2c exhibited punctum distribution and focal accumulation induced by sporelings in eps ( Fig. 8E ), but it did not confer susceptibility in eps3m ( Fig. 8B,C ), supporting the role of the C-terminus of MLO2 in focal accumulation. It is also worth noting that MLO10, MLO7’s closest family member, displayed punctum distribution ( Fig. 7J,L ) but only allowed very limited PM growth in the best-case scenario ( Fig. 7K ). In synergid cells, MLO10, but not MLO8, could restore fertility of mlo7 ( Jones et al., 2017 ), indicating functional diversification among these three clade III family members. Given this complexity, and the lack of localization data for MLO3, MLO4 and MLO5 in Arabidopsis leaf epidermal cells, it is difficult to infer which sequence features or polymorphisms underlie MLOs’ contrasting molecular functions as reflected by permitting PM pathogenesis and displaying signal-induced relocalization. Regulation of MLO2’s focal accumulation at the PPM Regarding PM penetration-induced MLO2 relocalization to the PPM ( Fig. 6 ), a key question is what specific signal(s) triggers its polarizing trafficking and how this process is regulated. Given that MLO2’s CaMBD-containing cytoplasmic C-terminus directs its focal accumulation, it is conceivable that changes in cytosolic calcium concentration—triggered by calcium influxes during fungal penetration of the host cell wall or plasma membrane—could induce MLO2-calmodulin binding or dissociation, thereby triggering MLO2’s relocalization to the PPM. Such a mechanism would imply that changes of cytosolic calcium levels differentially impact MLOs residing in different subcellular compartments (e.g., MLO2 mostly at the ER/Golgi versus MLO1 at the plasma membrane), likely due to structural differences in their C-termini and specific calmodulins they interact with. In this context, it is worth noting that MLO1n-2c remained its punctum distribution in leaf epidermal cells of eps3m insulted by sporelings ( Fig. 8C ). However, MLO1n-2c were relocalized to the PPM in eps ( Fig. 8G ), suggesting that host cell-wall or plasma membrane disruption induced by successful host entry triggers PPM-oriented trafficking pathway, resulting in dispatch of ER/Golgi-localized MLO2 and the non-functional MLO1n-2c to the PPM. Because successful host entry requires functional MLO2, this would imply a positive feedback mechanism for MLO2’s focal accumulation at the PPM. Without functional plasma membrane-localized MLO2 to initiate the ER/Golgi→ PPM-directed trafficking, MLO1n-2c would remain sequestered at the ER/Golgi. How MLO2’s ER/Golgi → PPM trafficking polarity is established remains unknown. Phosphatidylinositol-4,5-biphosphate [PI(4,5)P 2 ] is a lipid messenger that plays crucial role in polarized trafficking, guiding vesicle and protein movement to specific subcellular locations in both animals and plants ( Mei et al., 2012 ; Thapa and Anderson, 2012 ). Qin et al. reported that disruptive mutations in PIP5K1 and PIP5K2 significantly reduced MLO2-GFP’s focal accumulation at the fungal penetrations site ( Qin et al., 2020 ). Because PIP5Ks catalyze the phosphorylation of phosphatidylinositol 4-phosphate [PI(4)P] to produce PI(4,5)P 2 , this observation suggests that PI(4,5)P 2 may serve as a trafficking cue in directing MLO2 to the PPM. Unfortunately, results from our genetic study showed that both MLO2-GFP’s focal accumulation at the PPM and MLO2-mediated susceptibility to Gc UCSC1 remained unchanged in the absence of PIP5K1 and PIP5K2 ( Fig. 10 ). This suggests that PI(4,5)P 2 unlikely serves as a trafficking cue for MLO2’s enrichment at the PPM. FER is required for MLO7’s relocalization from Golgi bodies to the filiform apparatus in synergid cells ( Jones et al., 2017 ; Ju et al., 2021 ). However, our genetic data indicate that FER and probably five other CrRLK1L family members are dispensable for MLO2’s focal accumulation and its-mediated PM pathogenesis ( Fig. 9 ). This suggests that either there is deeper functional redundancy among the 17 CrRLK1L family members ( Lindner et al., 2012 ) or a CrRLK1L-independent mechanism distinct from that employed in synergid cells for relocalization of MLO7 is engaged for the regulation of MLO2’s re-localization to the PPM in leaf epidermal cells. In conclusion, data from this study definitively demonstrate that MLO2, MLO6, MLO12 are bona fide susceptibility factors of PM fungi. MLO2 (and, by inference, clade IV and V MLOs) appears to adopt a bipartite functional configuration: while the N-terminal 7TM portion executes a specific cellular function, such as cell wall and/or plasma membrane sealing and stabilization, the CaMBD-containing C-terminus orchestrates its spatiotemporal activity via signal-induced re-localization and enrichment. Additionally, our demonstration of the protein dosage effect for MLO2 in mediating susceptibility points to the potential for engineering PM “resistance” without severe early leaf senescence by downregulation of target MLOs via virus-induced gene silencing (VIGS) or promoter editing. Future investigations will focus on elucidating how clade V MLOs contribute to the sealing and stabilization of the plasma membrane–penetration peg–haustorial neckband junction, thereby accommodating haustorium differentiation and PM pathogenesis. Materials and Methods Plant lines and growth conditions All mutants used in this study were in the Arabidopsis thaliana accession Col-0 background. Mutants eds1-2 ( Bartsch et al., 2006 ), pad4-1 ( Jirage et al., 1999 ) and sid2-2 ( Wildermuth et al., 2001 ) have been previously described. The triple mutant eds1-2/pad4-1/sid2-2 mutant was generated by genetic crosses and identified by PCR genotyping as previously described ( Zhang et al., 2018 ). The mlo2-5/mlo6-2/mlo12-1 triple mutant was previously described ( Consonni et al., 2006 ). Seeds were sown in SunGro Horticulture (Agawam, MA, U.S.) and cold treated (4 ℃ for 2 days) before moving to growth chambers. Seedlings were transplanted and kept growing under 22 ℃, 75% relative humidity, short day (8 h light at ∼125 μmol m-2 s-1, 16 h dark) conditions for up to 14 weeks before use. EMS mutagenesis of seeds and mutant screening About 10,000 eps seeds (approx. 200 mg) were placed in a 250 ml glass flask. 15 mL dH2O and 30 μL 0.2% EMS were added and the seeds were shaken overnight. After washing with dH 2 O, seeds were blotted dry and mixed with 250 g fine sand and aliquoted into about 50 parts. Each aliquot of ∼5 g sand with seeds was sown evenly in SunGro Horticulture (Agawam, MA, U.S.) in 50 flats. Seedling were grown in a greenhouse for about two months till maturity. Seeds from 30-90 M1 plants were collected to make one M1 seed pool. A total of 102 M1 seed pools were obtained. For mutant screening, about 150-450 M2 plants per pool were prepared in one or two flats for inoculation with Gc UCSC1. Putative cipi mutants were transplanted into individual pots for further growth till maturity. Pathogen Infection, Disease Phenotyping, and Quantification The Arabidopsis-adapted powdery mildew isolate Golovinomyces cichoracearum ( Gc ) UCSC1 was maintained on live Col-0 or eds1-2 plants. Inoculation, visual scoring of disease reaction phenotypes and spore quantification were done as previously described (Xiao et al., 2005). Mapping of causal mutations Bulked segregant pool-genome sequencing was used to identify candidate causal mutations. cipi2 and cipi3 mutants were crossed with the eps parental line, and their corresponding F2 segregating populations were inoculated with Gc UCSC1. Genomic DNA was isolated from the mixture of the leaf tissues harvested from 65 or more F2 individuals showing the cipi phenotypes using NucleoSpin Plant II kit (MACHEREY-NAGEL, #740770.50). About 2 μg of each DNA sample was sent to BGI (Beijing Genomics Institute, Shenzhen, China) for deep sequencing using Illumina Hiseq 2000 platform at an average coverage of ∼50x with 100 bp paired end reads. The subsequent sequence analysis was done according to the method previously described ( Wang et al., 2018 ). Briefly, sequencing reads were mapped against the TAIR10 Arabidopsis reference genome using Bowtie ( Langmead et al., 2009 ) and variants were called by SAMtools ( Li et al., 2009 ). Only G to A and C to T conversions predominantly caused by EMS mutagenesis were picked for further analysis. The effect of each EMS-induced SNP (single-nucleotide polymorphism) on the corresponding gene was annotated using snpEffect ( Cingolani et al., 2012 ). The effects of the SNPs were classified into three categories: very high (stop gained, splice site donor, splice site acceptor), high (non-synonymous coding, start gained, stop lost), and other (intergenic, intron, 3’ UTR, 5’ UTR, synonymous coding). DNA constructs and Arabidopsis transformation The pK7FWG2 plasmid ( Karimi et al., 2002 ) was used for cloning of all MLO genes in translational fusion with eGFP. To replace the original 35S promoter with the MLO2 native promoter (pMLO2), restriction enzymes Xba I and Bam HI were used to linearize the plasmid, and ∼2 Kb pMLO2 fragment amplified from genomic DNA was inserted, leading to a pMLO2::ccdB-eGFP cassette used for cloning MLOx-eGFP fusion genes under control of the MLO2 promoter. To create various MLO expression constructs, corresponding MLO genes were first cloned into the pENTR/D-TOPO vector via TOPO cloning ( https://www.thermofisher.com/us/en/home/life-science/cloning/topo.html ). Then, the MLO genes were shuttled to the binary vector containing pMLO2::ccdB-eGFP via Gateway LR reaction. All vectors are verified by Sanger sequencing. Amplification of fragments for creation of chimeric MLOs was done using primers in Supplementary Table S1 by overlap-extension PCR. Specifically, the two fragments were amplified from Col-0 cDNA with overlapped chimeric primers. The two products were then mixed together at the same molar concentration and served as template for amplification of the full-length chimeric gene. All vectors are verified by Sanger sequencing. All binary vectors containing the MLOx-eGFP constructs were transfected into A. tumefaciens GV3101. Arabidopsis transformation was conducted following the floral dipping protocol described previously ( Clough and Bent, 1998 ). Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves N. benthamiana plants were grown in a growth chamber under 22 ℃, 75% relative humidity, long day (16 h light at ∼125 μmol m −2 s −1 , 8h dark) conditions for four weeks, and then were moved to short day condition (8 h light at ∼125 μmol m −2 s −1 , 16h dark) for 1 week before agroinfiltration. A. tumefaciens GV3101 cells containing corresponding vector(s) were suspended in infiltration buffer [10 mM MgCl 2 , 10 mM MES (pH 5.6), and 200 μM acetosyringone] to a final OD 600 value of 0.4-0.6. The agrobacterium suspension was incubated in dark for 2 h before infiltration into N. benthamiana leaves using a blunt syringe. The agroinfiltrated leaves were examined for MLOx-eGFP expression by confocal microscopy following the method described below. Confocal microscopy The expression and localization of the MLOx-eGFP fusion proteins were examined by confocal microscopy using a Zeiss LSM710 microscope. Saturated pixels are intentionally avoided. Confocal images were post-processed using ZEN software (Carl Zeiss, 2009 edition) with global linear adjustments applied consistently and in accordance with academic best practices for image processing. Detection of H 2 O 2 Accumulation and cell death DAB (3,3’-diaminobenzidine) staining was used to detect in situ H 2 O 2 production and accumulation while trypan blue staining was used to detect dead or dying cells as well as fungal structures in leaves. These methods were previously described ( Xiao et al., 2003 ). Quantitative RT-qPCR analysis Three leaf samples of seven-week-old plants (∼100 mg) per genotype were harvested before and at 0 hpi, 6 hpi, 12 hpi and 48 hpi after Gc UCSC1 infection. Total RNA was extracted using TRIzol® Reagent and reverse transcribed into cDNA using SuperScriptTM III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific Inc.). For each experiment, qRT-PCR was performed with three biological replicates per treatment and three technical replicates per sample using the Applied Biosystems 7300 Real-Time PCR System with SYBRTM Green PCR Master Mix (Thermo Fisher Scientific Inc.). The transcript levels of the target genes were normalized to that of UBC9 (Ubiquitin conjugating enzyme 9, AT4G27960). Data was analyzed using the Applied Biosystems 7300 Real-Time PCR System Software and the comparative ΔCt method. Primers used for qRT-PCR are listed in Supplementary Table S2 . Western blot Leaf tissue of 150 mg per sample was frozen in liquid nitrogen and grounded into fine powder. Two volume (w/v) Ripa buffer containing 100 μM PMSF, 1 x protease inhibitor, and 100 mM DTT was added to the frozen sample and then vortexed for homogenization. Samples were incubated at 95 ℃ for 5 min in 1 x SDS sample buffer, then centrifuged at 12000 g for 10 min. Samples were loaded to 4-12% Bis-Tris SurePAGE™ gel (GenScript #M00652) for electrophoresis following manufacturer’s instruction. Membrane transfer was conducted by 100 V for 2 h. The membrane was blocked with 5% BSA in TBST for 1h and then incubated with anti-GFP antibody (ab290, Abcam) overnight at 4 ℃. After 3×10 min washing with TBST, the membrane was loaded with secondary antibody (and incubated at room temperature for 1 h with shaking, followed by 3×10 min wash. The signal was generated by Clarity Western ECL Substrate (BIO-RAD ##1705061) and imaged with BIO-RAD ChemiDoc Imaging System. CRISPR/Cas9 targeted mutagenesis Two CRISPR/Cas9 genome editing systems were used for targeted mutagenesis of genes of interest in this study and all related plasmids were purchased from Addgene ( https://www.addgene.org ). The first utilizes an Arabidopsis egg cell-specific promoter to drive the expression of Cas9 ( Wang et al., 2015 ). This system was used to knockout MLO2 , MLO6 and MLO12 . The other system was developed for multiplexed CRISPR ( Stuttmann et al., 2021 ). It utilizes an intronized Cas9 to improve Cas9 expression and editing efficiency. This system was used to knockout FER and its other CrRLKL1 family members, as well as PIP5K1 and PIP5K2 . The pDGE347 binary destination vector was used for cloning the guide RNA constructs. All recombinant plasmids containing the guide RNA cassettes were confirmed by sequencing. All guide RNA sequences were listed in Supplementary Table S3 . Genotyping of mutants and transgenic lines All primers used for genotyping are listed in Supplementary Table S4 . To detect the mlo2 allele from cipi2 , the fragment containing the mutation was amplified by MLO2-e9F/MLO2-e11R followed by Mwo I digestion (New England Biolabs, R0573S). The wild type allele is cut into two smaller fragments (161+242 bp), while the cipi2 mutant allele is intact (403 bp). Similarly, to detect the mlo2 allele from cipi3 , the fragment containing the mutation was amplified by MLO2-e2F/MLO2-e3R followed by Hind III digestion (New England Biolabs, R3104S). While the wild type allele is intact (341 bp), the cipi3 mutant allele is digested into two fragments (179+162 bp). The remaining mlo2 alleles cipi11 , cipi12 , and cipi15 were amplified with MLO2e2F/MLO2-e3R, MLO2-e1F/MLO2-e3R, and MLO2-e6F/MLO2-e8R, respectively, followed by sequencing to detect the respective mutations. To detect mutations in FER and its family members, and in PIP5K1 and PIP5K2 , the guide RNA target regions were amplified with gene-specific primers (listed in Supplementary Table S4 ). PCR products were Sanger-sequenced. Benchling ( https://benchling.com/ ), a cloud-based platform for molecular biology data analysis, was used to make sequence alignments for identification of indels. Accession numbers DNA Sequences of the genes studied in this study can be found in the National Center for Biotechnology Information (NCBI) or The Arabidopsis Information Resource under the following gene ID numbers: AT4G02600 (Arabidopsis MLO1 ), AT1G11310 (Arabidopsis MLO2 ), AT3G45290 (Arabidopsis MLO3 ), AT1G11000 (Arabidopsis MLO4 ), AT2G33670 (Arabidopsis MLO5 ), AT1G61560 (Arabidopsis MLO6 ), AT2G17430 (Arabidopsis MLO7 ), AT5G65970 (Arabidopsis MLO10 ), AT5G53760 (Arabidopsis MLO11 ), AT2G39200 (Arabidopsis MLO12 ), AT2G19190 (Arabidopsis FRK1 ), AT5G44420 (Arabidopsis PDF1.2 ), AT5G14930 (Arabidopsis SAG101 ), AT2G14610 (Arabidopsis PR1 ), At1g21980 (Arabidopsis PIP5K1 ), At1g77740 (Arabidopsis PIP5K2 ), At5g38990 (Arabidopsis MDS1 ), At5g59700 (Arabidopsis ANJ ), At5g54380 (Arabidopsis THE1 ), At1g30570 (Arabidopsis HERK2 ), At3g51550 (Arabidopsis FER ), and unmade CrRLK1L family members, At2g23200, At2g39360, and At5g24010. Supplementary data Download figure Open in new tab Supplementary Figure S1. cipi3 exhibits strong “resistance” to Golovinomyces cichoracearum ( Gc ) UCSC1. (A) Eight-week-old plants of the indicated genotypes were infected with Gc UCSC1. Photos were taken at 10 dpi. Note the trichome-based infection in cipi3 . (B) Reduced susceptibility of cipi3 compared to Col-0 wild-type and the eps ( eds1/pad4/sid2 ) parental line as measure by total number of spores per mg infected leaves at 12 dpi. Different letters indicate statistically significant differences ( P <0.001) between the three lines, as determined by multiple comparisons using one-way ANOVA, followed by Tukey’s HSD test. This experiment was repeated once with similar results. Download figure Open in new tab Supplementary Figure S2. Predicted and sequence-confirmed mis-splicing of MLO2 mRNA in the cipi2 and cipi3 mutants. Shown are the exon-intron boundaries and the genomic DNA ( A,C ) and predicted and sequence-confirmed cDNAs ( B,D ) reflecting mRNAs resulted from mRNA splicing in wild-type and the mutation regions of cipi2 ( A,B ) and cipi3 ( C,D ). The nucleotides of the introns are in lower case. The splicing motifs are in highlighted in blue. The vertical bar indicates the splice boundary after removal of the intron in the mRNA. Arrows indicate the C -to-T mutation in cipi2 ( B ) or G-to-A the mutation in cipi3 ( D ) confirmed by the respective Sanger sequencing chromatographs. Download figure Open in new tab Supplementary Figure S3. CRISPR-targeted mutagenesis of MLO6 and MLO12 . (A) MLO6’s gene structure with the protospacer and PAM sequence marked. (B) Three independent lines with indels in MLO6 and the position of the premature stop codon marked with red hexagons. (C) MLO12’s gene structure with the protospacer and PAM sequence marked. (D) Three independent lines with indels in MLO12 and the position of premature stop codon marked with red hexagons. Download figure Open in new tab Supplementary Figure S4. No haustorium formation in pavement cells of eps3m . Plants of eps and eps3m were inoculated with Gc UCSC1. At 6 dpi, infected leaves were gently brushed using a fine brush under tap water for 30 seconds to completely remove fungal sporelings and mycelia on the leaf surface and then subjected to trypan blue staining. Dark blue-stained haustoria are visible in the pavement cells of eps but not found in those of eps3m . Instead, small blue dots (arrows) likely representing penetration sites were visible in pavement cells of eps3m . In contrast, haustoria can be found in infected trichome cells of eps and eps3m (inset). Download figure Open in new tab Supplementary Figure S5. No activation of PTI and ETI marker genes in eps3m . Six-week-old, short-day grown plants of the four indicated genotypes were inoculated with Gc UCSC1. Inoculated leaves were collected at the indicated four timepoints and subjected to qRT-qPCR analysis to measure expression of four indicated marker genes. The control group is defined as Col-0, 0h. All gene groups pass ANOVA with p <0.05. Post-hoc analyses (multiple comparisons) are conducted through T -test adjusted by Benjamini-Hochberg FDR procedure. * p <0.05, ** p <0.01, *** p <0.001”. (A) Expression of FRK1 , reporting activation of PTI. (B) Expression of PR1 , reporting activation of ETI. (C) Expression of PDF1.2 , reporting activation of the Jasmonic acid- and ethylene-dependent defenses. (D) Expression of SAG101 , reporting the onset of leaf senescence. This experiment was repeated twice with similar results. Download figure Open in new tab Download figure Open in new tab Supplementary Figure S6. MLO2-GFP is localized to the peri-penetration peg membranous space (PPM) next to the haustorial neck. Plants of eps3m expressing MLO2-GFP and RPW8.2-RFP were inoculated with Gc UCSC1. Infected leaves were subjected to confocal microscopy at 2 -3 dpi. (A-C) Three z-stack (3-5) projected confocal images with the GFP and RFP individual channels. Note, the merged images are shown in Fig. 7F -H . Bar=10μm. (D-G) Additional four confocal images from 3-5 z-stack projection. Bar=10μm. Download figure Open in new tab Supplementary Figure S7. Expression of HvMLO1-GFP, MLO6-GFP or MLO7-GFP in eps3m partially restored susceptibility to Gc UCSC1. (A) Representative T1 plants of eps3m transgenic for 35S::HvMLO1 infected with Gc UCSC1 at 12 dpi. Note, three T1 plants were moderately susceptible (red stars) while one was weakly susceptible (yellow star) compared to eps plants. (B,C) Representative photos of the indicated transgenic lines infected with Gc UCSC1 at 11 dpi (B) and their levels of susceptibility (C). Asterisk indicates significant difference ( p <0.001; unpaired Student’s t -test). (D) Infection phenotypes of the T2 progenies of two eps3m lines transgenic for pMLO2::MLO7-GFP . Photos were taken at 11 dpi with Gc UCSC1. Download figure Open in new tab Supplementary Figure S8. Selection and cloning of seven MLO family members from five different clades for ectopic expression in leaves of eps3m by the MLO2 promoter. (A) A phylogenetic tree of the Arabidopsis MLO family plus barley and wheat clade IV MLO1 constructed based on deduced amino acid sequences using MEGA12 [Kumar S., Stecher G., Suleski M., Sanderford M., Sharma S., and Tamura K. (2024). Molecular Evolutionary Genetics Analysis Version 12 for adaptive and green computing. Molecular Biology and Evolution 41:1-9]. Bold-faced are MLO family members (belonging to different clades) that were subjected to expression and localization analyses. (B) Schematic showing the binary vector for expressing the eight indicated MLO genes from the MLO2 promoter. Download figure Open in new tab Supplementary Figure S9. MLO1-GFP and MLO11-GFP are localized to the plasma membrane. Leaves of eps3m transgenic plants expressing MLO1-GFP ( A ) or MLO11-GFP ( B ) from the MLO2 promoter were stained with 20 µM FM4-64 for 15 min before subjected to confocal imaging. Shown are representative z-stack confocal images projected from 3-5 thin optical sections. Download figure Open in new tab Supplementary Figure S10. MLO3-GFP exhibits ER-localization in leaf epidermal cells of N. benthamiana . Agrobacterium cells harboring pMLO2::MLO3-GFP were mixed in equal concentration (OD 600 =0.5) with those harboring the ER marker 35S::HEDL-mCherry ( A ) or the Golgi marker 35S::Man1-mCherry ( B ). The mixtures were infiltrated into leaves of N. benthamiana . Confocal images were acquired at 2 days after agroinfiltration. Shown are Z-stack projections of 3-5 optical sections. Download figure Open in new tab Supplementary Figure S11. MLO4-GFP exhibits partial ER- and partial Golgi-localization in leaf epidermal cells of N. benthamiana . Agrobacterium cells harboring pMLO2::MLO4-GFP were mixed in equal concentration (OD 600 =0.5) with those harboring the ER marker 35S::HEDL-mCherry (A) or the Golgi marker 35S::Man1-mCherry (B). The mixtures were infiltrated into leaves of N. benthamiana . Confocal images were acquired at 2 days after agroinfiltration. Shown are Z-stack projections of 3-5 optical sections. Download figure Open in new tab Supplementary Figure S12. MLO5-GFP exhibits partial ER- and partial Golgi-localization in leaf epidermal cells of N. benthamiana . Agrobacterium cells harboring pMLO2::MLO5-GFP were mixed in equal concentration (OD 600 =0.5) with those harboring the ER marker 35S::HEDL-mCherry ( A ) or the Golgi marker 35S::Man1-mCherry ( B ). The mixtures were infiltrated into leaves of N. benthamiana . Confocal images were acquired at 2 days after agroinfiltration. Shown are Z-stack projections of 3-5 optical sections. Download figure Open in new tab Supplementary Figure S13. CRISPR-targeted mutagenesis of FER . The CRISPR construct was introduced in eps (A) and eps3m expressing MLO2-GFP and RPW8.2-RFP (B). (A) Sanger sequencing chromatograms of the sgRNA target regions of three independent T1 lines with a compact rosette in the eds1/pad4/sid2 ( eps ) triple mutant background. (B) Sanger sequencing chromatograms of the sgRNA target regions of three independent T1 lines with a compact rosette in the background of eds1/pad4/sid2/mlo2/mlo6/mlo12 ( eps3m ) plants transgenic for pMLO2::MLO2-GFP and pRPW8.2::RPW8.2-RFP . Download figure Open in new tab Supplementary Figure S14. Multiplexed CRISPR targeting eight CrRLK1L family members. The multiplexed CRISPR construct was introduced into eps3m expressing MLO2-GFP and RPW8.2-RFP. Shown are the sequence alignments between the wild-type (Col-0) sequence and the Sanger sequencing chromatograms of the sgRNA-targeting regions of eight CrRLK1L genes in one mutant line (e2) exhibiting compact rosette and susceptibility phenotypes. Notably, six (highlighted in red) of the eight targeted CrRLK1L genes contain disruptive indels. Download figure Open in new tab Supplementary Figure S15. CRISPR-targeted mutagenesis of PIP5K1 and PIP5K2 . Shown are 42 T1 plants transgenic for a CRISPR construct targeting PIP5K1 and PIP5K2 in eps3m expressing MLO2-GFP and RPW8.2-RFP at 10 dpi with Gc UCSC1. Note, 17 of the 42 T1 plants exhibited greatly reduced stature and susceptibility to Gc UCSC1. View this table: View inline View popup Download powerpoint Supplementary Table S1. Primers used for cloning MLO genes. View this table: View inline View popup Download powerpoint Supplementary Table S2. Primers used for gRT-qPCR. View this table: View inline View popup Download powerpoint Supplementary Table S3. Small guide RNAs used for CRISPR/Cas9-targeted mutagenesis. View this table: View inline View popup Supplementary Table S4. Primers used for genotyping or sequencing. AUTHOR CONTRIBUTIONS S.X., C-I.W., and Q.Z. designed and initiated the project; Q.Z. identified the mlo2 mutants, D.B., Q.Z., S.X., Y.W., P.L., M.P., C.Z., A.H., J.Z., performed various experiments; R.P. and S.K. provided research materials and improve the manuscript; L.S. and P.H. helped with mutant characterization; D.B., Q.Z. and S.X. analyzed data and wrote the manuscript with help from other coauthors. DECLARATION OF INTERESTS The authors declare no competing interests. ACKNOWLEDGMENTS We thank Frank Coker and Caroline Hooks for their assistance in maintenance of plant growth facility a critically reading the article. This work was supported the National Sciences Foundation (IOS-1901566; IOS-2224203) to S.X. Funder Information Declared National Science Foundation, https://ror.org/021nxhr62 , IOS-1901566 , IOS-2224203 Footnotes In the revised version of the manuscript, the Supplementary Figures were added; the original Figure 5 was converted to Supplementary Figure S5; Figure 6 (now Figure 5) was slightly modified; Data description was changed accordingly. REFERENCES ↵ Acevedo-Garcia , J. , Kusch , S. , and Panstruga , R . ( 2014 ). Magical mystery tour: MLO proteins in plant immunity and beyond . New Phytol 204 , 273 – 281 . 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Share Clade V MLO proteins are bona fide host susceptibility factors required for powdery mildew pathogenesis in Arabidopsis David Bloodgood , Qiong Zhang , Pai Li , Ying Wu , Michael Pan , Christina Zhou , Apsen Hsu , Jun Zhang , Ralph Panstruga , Sharon Kessler , Ping He , Libo Shan , Chang-I Wei , Shunyuan Xiao bioRxiv 2025.06.18.660284; doi: https://doi.org/10.1101/2025.06.18.660284 Share This Article: Copy Citation Tools Clade V MLO proteins are bona fide host susceptibility factors required for powdery mildew pathogenesis in Arabidopsis David Bloodgood , Qiong Zhang , Pai Li , Ying Wu , Michael Pan , Christina Zhou , Apsen Hsu , Jun Zhang , Ralph Panstruga , Sharon Kessler , Ping He , Libo Shan , Chang-I Wei , Shunyuan Xiao bioRxiv 2025.06.18.660284; doi: https://doi.org/10.1101/2025.06.18.660284 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7640) Biochemistry (17706) Bioengineering (13902) Bioinformatics (41978) Biophysics (21465) Cancer Biology (18611) Cell Biology (25528) Clinical Trials (138) Developmental Biology (13387) Ecology (19920) Epidemiology (2067) Evolutionary Biology (24332) Genetics (15615) Genomics (22519) Immunology (17747) Microbiology (40424) Molecular Biology (17194) Neuroscience (88662) Paleontology (667) Pathology (2838) Pharmacology and Toxicology (4827) Physiology (7650) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9826) Zoology (2271)