CRISPR/SpCas9-mediated KO of epigenetically active MORC proteins increases barley resistance to Bipolaris spot blotch and Fusarium root rot

Microrchidia (MORC) proteins are fundamental regulators of genome stabilization, chromatin remodeling and gene expression in both mammals and plants. In Arabidopsis, their activity is linked to the RNA-directed DNA methylation (RdDM) pathway, which utilizes small RNAs (sRNAs) to influence the rate of DNA methylation and chromatin compaction and thus gene expression. In barley, there are a total of seven members of the MORC family, and recent advances showed that HvMORC1 and HvMORC6a also interact with components of the RdDM pathway. CRISPR/SpCas9-mediated single and double knock-out mutants showed de-repression of transposable elements (TEs) and pathogenesis-related (PR) genes and interestingly increased resistance to both biotrophic and necrotrophic plant pathogenic fungi. In this study, we further demonstrate the requirement of MORC proteins in the resistance against two devastating cereal diseases, Bipolaris spot blotch, caused by Bipolaris sorokiniana and Fusarium root rot, caused by Fusarium graminearum.


Introduction
In eukaryotes, transcriptional gene silencing (TGS) results in decreased RNA synthesis by establishing and maintaining DNA methylation through the RNA-directed DNA methylation (RdDM) pathway (Law and Jacobsen 2010;Erdmann and Picard 2020). In Arabidopsis, several members of the Microrchidia (MORC) protein family (AtMORC1 to AtMORC7) are RdDM downstream players involved in repression of DNA methylated genes as well as transposable elements (TEs) by increasing chromatin compaction rate (Lorković et al. 2012;Moissiard et al. 2012Moissiard et al. , 2014Brabbs et al. 2013;Liu et al. 2014Liu et al. , 2016Harris et al. 2016;Jing et al. 2016;Xue et al. 2021). Several studies have shown that MORC proteins play a role in plant defense, but it is highly dependent on the plant species. In Arabidopsis and potato, MORC proteins enhance resistance to pathogens, while in barley, tobacco and tomato, they negatively affect plant immunity (Kang et al. 2008(Kang et al. , 2010(Kang et al. , 2012Langen et al. 2014;Manosalva et al. 2015;Kumar et al. 2018). Thus, in clear contrast to Arabidopsis mutants that show reduced expression of pathogenesis-related genes (PRs), knock-down (KD) and knock-out (KO) mutants of barley MORC genes show enhanced PRs expression. Moreover, HvMORC1, HvMORC2 and HvMORC6a proteins also play a crucial role in maintaining genome stability by suppressing TEs (Langen et al. 2014;Kumar et al. 2018), and forming nucleocytoplasmic homo-/heteromeric MORC complexes that contain additional components of the RdDM gene silencing machinery . As a consequence, mutations in HvMORC1 and HvMORC6a resulted in de-repression 1 3 of TEs and were associated with increased disease resistance to powdery mildew caused by Blumeria graminis f.sp. hordei (Bgh) and Fusarium leaf spot. It is also noteworthy that MORC mutants exhibit reduced leaf and root development in addition to their effects on the plant immune system ).
In the present study, we have extended our analysis on the role of barley MORC proteins in RdDM-mediated epigenetic regulation of disease resistance, using Bipolaris sorokiniana (Bs) (teleomorph Cochliobolus sativus) and Fusarium root rot (FRR) caused by Fg as study cases as they are two major cereal pathogens of global importance. Bs is the causative agent of cereal spot blotch disease, which severely limits grain production in warm, humid South Asian countries, as well as in Canada, the USA, Brazil and Australia, resulting in significant yield losses (Kumar et al. 2002;Singh et al. 2015;Gupta et al. 2018). Fusarium fungi, on the other hand, are devastating plant pathogens of wheat and barley that are widespread worldwide causing Fusarium head blight (FHB), Fusarium crown rot (FCR) and Fusarium root rot (FRR) (Hollaway et al. 2013;Balmas et al. 2015). They also contaminate the grain with mycotoxins and thus decrease grain quality and availability (Gaffar et al. 2019). We show here that barley MORC single and double mutants generated with CRISPR/SpCas9 exhibit increased resistance to Bipolaris spot blotch and FRR.

Plant inoculation
Bs conidia were applied on whole ten-day-old seedlings with a spray brush (20 × 10 4 conidia ml −1 ) in 0.002% Tween water (v/v). Mock inoculated control plants were sprayed with Tween water only. After inoculation, the plants were placed in a transparent plastic box with moist paper towels and closed with a lid to ensure > 95% relative humidity. After five days, the first leaf of each plant was cut off, placed on (1% w/v) agar plate, photographed, and the black Bs lesions were subsequently counted. For Fg root rot analysis, threeday-old barley seedlings were placed in glass jars and roots submerged with 15 × 10 4 conidia ml −1 in Tween water for 90 min. The control group was treated with Tween water only. Subsequently, the plants were wrapped in moist sterile filter paper and placed in 50 mL falcon tubes for seven days under 22 °C, 16/8 h light. Infection was monitored regularly, and fungal growth was assessed seven days post-inoculation (dpi).

Real-time PCR detection for quantification of infection levels
The same plant material was used for quantitative analysis as for symptom assessment. Seven-day-old infected and non-infected leaves with Bs were crushed in liquid nitrogen, and DNA was extracted using a DNA extraction kit (Qiagen, Hilden, Germany). Total fungal/plant DNA ratio was quantified by quantitative real-time (qPCR) using fungal Glyceraldehyde-3-phosphate dehydrogenase (BsGPD) primers (Table 1) Galli et al. (2021). For quantification of Fg DNA, root samples were ultrasonically washed three times in a water bath prior to DNA extraction. Quantification by qPCR was performed with fungal Elongation factor-1 α (FgEF-1a) primers (Table 1) normalized to plant Ubiquitin (HvUbi) as described in Galli et al. (2021). Statistical tests used in this study (Student's t test, Kruskal-Wallis test with multiple comparisons and ANOVA with post hoc Tukey HSD test) were selected after analysis of the normal distribution and homogeneity of variances in the different groups. All biological assays were repeated at least twice with similar results.
Compared to leaf-infecting pathogens, fewer studies focus on root pathogens due to the difficulty in observing the infection process. F. graminearum is not only a specialized pathogen of the aerial parts but can also cause tremendous damage by root rot (Lanoue et al. 2010;Kazan and Gardiner 2018). We addressed the question of whether KO of MORCs also leads to higher resistance to FRR. To this end, seedlings were dip-inoculated in Fg conidia, and infection was assessed by quantifying the fungal DNA by qPCR at 7 dpi. Significant reduction in fungal growth was observed in all mutants as compared to HvWT (Δhvmorc1 L3: 2.1-fold decrease; Δhvmorc6a L9: 1.9-fold decrease; Δhvmorc6a L16: 1.7-fold decrease; Δhvmorc1/6a L4: 1.5-fold decrease; Δhvmorc1/6a L5: 1.4-fold decrease; Student's t test versus HvWT and α = 0.05, ANOVA with Tukey test; Fig. 2).

Epigenetic mechanisms in plant defense
MORC proteins were first identified in mice as important regulators of spermatogenesis and genital development (Watson et al. 1998;Inoue et al. 1999). They are also widespread among plants, where they bear highly conserved ATP binding motifs to their counterparts in animal phyla (Langen et al. 2014;Dong et al. 2018;Xue et al. 2021). Accumulating structural and biochemical evidence suggests that both plant and animal MORCs share many similar functions related to disease resistance and epigenetic control (for review see Li et al. 2013;Koch et al. 2017;Xue et al. 2021). Epigenetic gene regulation is fundamental for genome integrity, gene expression and the repression of TEs. The plant-specific RdDM pathway achieves stable epigenetic modification via both de novo and maintenance of DNA and chromatin methylation (Lister et al. 2008;Law and Jacobsen 2010;Du et al. 2015;Matzke et al. 2015;Erdmann and Picard 2020). Several studies have shown that epigenetic regulation fine-tunes the trade-off between disease resistance, yield and fitness (Dowen et al. 2012;Deng et al. 2017;Kumar et al. 2021). Here, we show that the CRISPR/SpCas9-generated KO mutations in HvMORC1 and HvMORC6a have an enhanced immune response to Bipolaris leaf spot blotch (Fig. 1a-c) and Fusarium root rot (Fig. 2). These results are consistent with previous reports showing that barley MORC mutants also have increased resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) fungal leaf pathogens (Langen et al. 2014;Kumar et al. 2018;Galli et al. 2021). Interestingly, the enhanced-defense phenotype of single versus double KO mutants was not additive. This finding is consistent with previous reports suggesting that HvMORC1 and HvMORC6a require each other's activity for immune function because they physically interact as a prerequisite for their action .
Given that the defense mechanisms involved in resistance to biotrophic, hemibiotrophic and necrotrophic pathogens often function antagonistically due to the antagonism mode of action inherent to the defense pathways associated with the plant hormones salicylic acid and jasmonic acid (Glazebrook 2005; Klessig et al. 2018), our finding is relevant in the plant pathology field: 1. The involvement of MORC proteins in plant defense is independent of the known hormone-driven antagonistic defense mechanisms; 2. epigenetic regulation of plant defense has so far been neglected in breeding because the mechanism is still unexplored and very complex. 3. Breeding strategies with a focus on epigenetics could have the advantage that they may avoid the hormonal antagonism in plant defense and thus work on a broader type of resistance.
In conclusion, we hypothesize that KO of HvMORC1 and HvMORC6a achieves higher immunity by weakening the recruitment of RdDM complexes to their site of action on barley DNA and chromatin. Plant TEs are silenced via the RdDM pathway, and methylation often extends to flanking genes (Cui and Cao 2014). HvMORC1 and HvMORC6a form distinct nucleo-cytoplasmic homo-/heteromers with Fig. 1 Response of SpCas9-induced barley mutants against Bipolaris sorokiniana (Bs), the causal agent of spot blotch disease. a Single T3 mutants Δhvmorc1 L3, Δhvmorc6a L9, Δhvmorc6a L16 and T3 double mutants Δhvmorc1/6a L4, Δhvmorc1/6a L5 show increased resistance to the hemibiotrophic fungus Bs KN2 as revealed by reduced numbers of necrotic lesions. Shown is the average number of Bs necrotic lesions on leaves (n = 8) at 5 dpi in two biological replicates. Comparisons between groups were performed via student's t test between HvWT and mutants; asterisks represent statistical difference of the groups against HvWT (Student's t test: *p < 0.05; **p < 0.01; ***p < 0.001). Comparisons between groups were per-formed via Kruskal-Wallis test with multiple comparisons. Letters represent statistical differences among all group means (α < 0.05). b Quantitative analysis of fungal infection as calculated as total amount of Bs DNA in barley leaves (7 dpi), based on the ratio of fungal Glyceraldehyde-3-phosphate dehydrogenase (BsGPD) calculated by qPCR. Bars represent the standard deviation (SD) of three technical repetitions; biological assay was repeated twice with similar results. Comparisons between groups were performed by ANOVA and Tukey's range test for multiple comparisons. Letters represent statistical differences among all group means (α < 0.05). c Leaf infection phenotypes after Bs spray inoculation (7 dpi) other HvMORCs and interact with components of the RdDM pathway . Our results are also consistent with the idea that in the absence of a pathogen barley MORCs interact with DNA-methylating proteins to repress TEs and favorite chromatin compaction of TEs` flanking regions, leading to the transcriptional repression of several genes, including PRs (Fig. 3). On the contrary, a compromised function of MORC proteins then explains higher disease resistance to various fungal pathogens (Fig. 1a-c and Fig. 2), constitutive higher TEs and PRs expression (even if no pathogen is present), and atypical plant growth Galli et al. 2021).
Importantly, we show that barley MORC-mediated epigenetic regulation fine-tunes disease resistance to a very broad range of fungal pathogens. Previous reports already showed a trade-off between MORC-dependent plant defense and Fig. 2 Response of SpCas9-induced barley mutants against Fusarium graminearum (Fg), the causal agent of FRR. Single T3 mutants Δhvmorc1 L3, Δhvmorc6a L9, Δhvmorc6a L16 and T3 double mutants Δhvmorc1/6a L4 and Δhvmorc1/6a L5 show increased root resistance to Fg as revealed by quantification of total Fg DNA. Whole roots of 3-day-old seedlings were dip-inoculated with 30 mL solution of Fg conidia (15 × 10 4 conidia mL −1 ) for 90 min. qPCR was used to measure the amount of Fg DNA at 7 dpi, calculated based on the ratio of fungal Elongation factor-1 α (FgEF-1a). Bars represent the SD of three technical repetitions; the biological assay was repeated twice with similar results. Asterisks represent statistical difference of the groups against HvWT (Student's t test: *p < 0.05; **p < 0.01; ***p < 0.001) Fig. 3 Simplified working model hypothesis of the role of barley MORC proteins in the regulation of TEs, defense genes and disease resistance. MORC protein complexes interact directly with other components of the RdDM pathway to initiate or maintain inhibitory methylation marks, leading to the formation of heterochromatin, silencing of TEs and potentially many defense genes in the environment (reduced disease resistance). The absence of MORCs then leads to relaxed chromatin, de-repression of various silenced TEs and many flanking genes, including PRs (increased disease resistance) growth , thus revealing possible gene candidates in a single cluster that have evolved opposing functions in disease resistance and plant fitness. In the future, the discovery of the effects of MORC on these important agronomic traits and the importance of the RdDM pathway in pathological processes may help to develop new breeding strategies for higher yielding and more resistant barley varieties.