Genome editing in plants using the compact editor CasΦ.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.

The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis.

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

DNA methylation-linked chromatin accessibility affects genomic architecture in Arabidopsis.

DNA methylation is a major epigenetic modification found across species and has a profound impact on many biological processes. However, its influence on chromatin accessibility and higher-order genome organization remains unclear, particularly in plants. Here, we present genome-wide chromatin accessibility profiles of 18 Arabidopsis mutants that are deficient in CG, CHG, or CHH DNA methylation. We find that DNA methylation in all three sequence contexts impacts chromatin accessibility in heterochromatin. Many chromatin regions maintain inaccessibility when DNA methylation is lost in only one or two sequence contexts, and signatures of accessibility are particularly affected when DNA methylation is reduced in all contexts, suggesting an interplay between different types of DNA methylation. In addition, we found that increased chromatin accessibility was not always accompanied by increased transcription, suggesting that DNA methylation can directly impact chromatin structure by other mechanisms. We also observed that an increase in chromatin accessibility was accompanied by enhanced long-range chromatin interactions. Together, these results provide a valuable resource for chromatin architecture and DNA methylation analyses and uncover a pivotal role for methylation in the maintenance of heterochromatin inaccessibility.

HAG Effector Evolution in Pyricularia Species and Plant Cell Death Suppression by HAG4.

Seventy host-adapted gene (HAG) effector family members from Pyricularia species are found in P. oryzae and three closely related species (isolates LS and 18-2 from an unknown Pyricularia sp., P. grisea, and P. pennisetigena) that share at least eight orthologous HAG family members with P. oryzae. The genome sequence of a more distantly related species, P. penniseti, lacks HAG genes, suggesting a time frame for the origin of the gene family in the genus. In P. oryzae, HAG4 is uniquely found in the genetic lineage that contains populations adapted to Setaria and Oryza hosts. We find a nearly identical HAG4 allele in a P. grisea isolate, suggesting transfer of HAG4 from P. grisea to P. oryzae. HAG4 encodes a suppressor of plant cell death. Yeast two-hybrid screens with several HAG genes independently identify common interacting clones from a rice complementary DNA library, suggesting conservation of protein surface motifs between HAG homologs with as little as 40% protein sequence identity. HAG family orthologs have diverged rapidly and HAG15 orthologs display unusually high rates of sequence divergence compared with adjacent genes suggesting gene-specific accelerated divergence. The sequence diversity of the HAG homologs in Pyricularia species provides a resource for examining mechanisms of gene family evolution and the relationship to structural and functional evolution of HAG effector family activity. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Evolution and Regulation of a Large Effector Family of Pyricularia oryzae.

Plant pathogen effectors play important roles in parasitism, including countering plant immunity. However, investigations of the emergence and diversification of fungal effectors across host-adapted populations has been limited. We previously identified a gene encoding a suppressor of plant cell death in Pyricularia oryzae (syn. Magnaporthe oryzae). Here, we report the gene is one of a 21-member gene family and we characterize sequence diversity in different populations. Within the rice pathogen population, nucleotide diversity is low, however; the majority of gene family members display presence-absence polymorphism or other null alleles. Gene family allelic diversity is greater between host-adapted populations and, thus, we named them host-adapted genes (HAGs). Multiple copies of HAGs were found in some genome assemblies and sequence divergence between the alleles in two cases suggested they were the result of repeat-induced point mutagenesis. Transfer of family members between populations and novel HAG haplotypes resulting from apparent recombination were observed. HAG family transcripts were induced in planta and a subset of HAGs are dependent on a key regulator of pathogenesis, PMK1. We also found differential intron splicing for some HAGs that would prevent ex planta protein expression. For some genes, spliced transcript was expressed in antiphase with an overlapping antisense transcript. Characterization of HAG expression patterns and allelic diversity reveal novel mechanisms for HAG regulation and mechanisms generating sequence diversity and novel allele combinations. This evidence of strong in planta-specific expression and selection operating on the HAG family is suggestive of a role in parasitism.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Carbon catabolite repressor MoCreA is required for the asexual development and pathogenicity of the rice blast fungus.

During the infection and colonization process, the rice blast fungus Magnaporthe oryzae faces various challenges from hostile environment, such as nutrient limitation and carbon stress, while carbon catabolite repression (CCR) mechanism would facilitate the fungus to shrewdly and efficiently utilize carbon nutrients under fickle nutritional conditions since it ensures the preferential utilization of most preferred carbon sources through repressing the expression of enzymes required for the utilization of less preferred carbon sources. Researches on M. oryzae CCR have made some progress, however the involved regulation mechanism is still largely obscured, especially, little is known about the key carbon catabolite repressor CreA. Here we identified and characterized the biological functions of the CreA homolog MoCreA in M. oryzae. MoCreA is constitutively expressed throughout all the life stages of the fungus, and it can shuttle between nucleus and cytoplasm which is induced by glucose. Following functional analyses of MoCreA suggested that it was required for the vegetative growth, conidiation, appressorium formation and pathogenicity of M. oryzae. Moreover, comparative transcriptomic analysis revealed that disruption of MoCreA resulted in the extensive gene expression variations, including a large number of carbon metabolism enzymes, transcription factors and pathogenicity-related genes. Taken together, our results demonstrated that, as a key regulator of CCR, MoCreA plays a vital role in precise regulation of the asexual development and pathogenicity of the rice blast fungus.

Bayogenin 3-O-cellobioside confers non-cultivar-specific defence against the rice blast fungus Pyricularia oryzae.

Rice cultivars from japonica and indica lineage possess differential resistance against blast fungus as a result of genetic divergence. Whether different rice cultivars also show distinct metabolomic changes in response to P. oryzae, and their role in host resistance, are poorly understood. Here, we examine the responses of six different rice cultivars from japonica and indica lineage challenged with P. oryzae. Both susceptible and resistant rice cultivars expressed several metabolites exclusively during P. oryzae infection, including the saponin Bayogenin 3-O-cellobioside. Bayogenin 3-O-cellobioside level in infected rice directly correlated with their resistant attributes. These findings reveal, for the first time to our knowledge that besides oat, other grass plants including rice produces protective saponins. Our study provides insight into the role of pathogen-mediated metabolomics reprogramming in host immunity. The correlation between Bayogenin 3-O-Cellobioside levels and blast resistance suggests that engineering saponin expression in cereal crops represents attractive and sustainable disease management.

Mechanistic insights into plant SUVH family H3K9 methyltransferases and their binding to context-biased non-CG DNA methylation.

DNA methylation functions in gene silencing and the maintenance of genome integrity. In plants, non-CG DNA methylation is linked through a self-reinforcing loop with histone 3 lysine 9 dimethylation (H3K9me2). The plant-specific SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) family H3K9 methyltransferases (MTases) bind to DNA methylation marks and catalyze H3K9 methylation. Here, we analyzed the structure and function of Arabidopsis thaliana SUVH6 to understand how this class of enzyme maintains methylation patterns in the genome. We reveal that SUVH6 has a distinct 5-methyl-dC (5mC) base-flipping mechanism involving a thumb loop element. Autoinhibition of H3 substrate entry is regulated by a SET domain loop, and a conformational transition in the post-SET domain upon cofactor binding may control catalysis. In vitro DNA binding and in vivo ChIP-seq data reveal that the different SUVH family H3K9 MTases have distinct DNA binding preferences, targeting H3K9 methylation to sites with different methylated DNA sequences, explaining the context biased non-CG DNA methylation in plants.

Genome Sequence Resource of Magnaporthe oryzae Laboratory Strain 2539.

Magnaporthe oryzae causes blast disease on more than 50 species of monocot plants, including important crops such as rice, millet, and most recently wheat. Additionally, it is an important model system for studying host-pathogen interaction. Here, we report a high-quality genome assembly and annotation of a laboratory strain 2539 of M. oryzae, which is a widely used progeny of a rice-infecting isolate and a grass-infecting isolate. The genome sequence of strain 2539 will be useful for studying the evolution, host adaption, and pathogenicity of M. oryzae, which will be beneficial for a better understanding of the mechanisms of host-pathogen interaction.

Emergence of a hybrid PKS-NRPS secondary metabolite cluster in a clonal population of the rice blast fungus Magnaporthe oryzae.

Secondary metabolites (SMs) are crucial for fungi and vary in function from beneficial antibiotics to pathogenicity factors. To generate diversified SMs that enable different functions, SM-coding regions rapidly evolve in fungal genomes. However, the driving force and genetic mechanism of fungal SM diversification in the context of host-pathogen interactions remain largely unknown. Previously, we grouped field populations of the rice blast fungus Magnaporthe oryzae (syn: Pyricularia oryzae) into three major globally distributed clades based on population genomic analyses. Here, we characterize a recent duplication of an avirulent gene-containing SM cluster, ACE1, in a clonal M. oryzae population (Clade 2). We demonstrate that the ACE1 cluster is specifically duplicated in Clade 2, a dominant clade in indica rice-growing areas. With long-read sequencing, we obtained chromosome-level genome sequences of four Clade 2 isolates, which displayed differences in genomic organization of the ACE1 duplication process. Comparative genomic analyses suggested that the original ACE1 cluster experienced frequent rearrangement in Clade 2 isolates and revealed that the new ACE1 cluster is located in a newly formed and transposable element-rich region. Taken together, these results highlight the frequent mutation and expansion of an avirulent gene-containing SM cluster through transposable element-mediated whole-cluster duplication in the context of host-pathogen interactions.

Whole genome and transcriptome analysis reveal adaptive strategies and pathogenesis of Calonectria pseudoreteaudii to Eucalyptus.

BACKGROUND: Leaf blight caused by Calonectria spp. is one of the most destructive diseases to affect Eucalyptus nurseries and plantations. These pathogens mainly attack Eucalyptus, a tree with a diversity of secondary metabolites employed as defense-related phytoalexins. To unravel the fungal adaptive mechanisms to various phytoalexins, we examined the genome of C. pseudoreteaudii, which is one of the most aggressive pathogens in southeast Asia. RESULTS: A 63.7 Mb genome with 14,355 coding genes of C. pseudoreteaudii were assembled. Genomic comparisons identified 1785 species-specific gene families in C. pseudoreteaudii. Most of them were not annotated and those annotated genes were enriched in peptidase activity, pathogenesis, oxidoreductase activity, etc. RNA-seq showed that 4425 genes were differentially expressed on the eucalyptus(the resistant cultivar E. grandis×E.camaldulensis M1) tissue induced medium. The annotation of GO term and KEGG pathway indicated that some of the differential expression genes were involved in detoxification and transportation, such as genes encoding ABC transporters, degrading enzymes of aromatic compounds and so on. CONCLUSIONS: Potential genomic determinants of phytoalexin detoxification were identified in C. pseudoreteaudii by comparison with 13 other fungi. This pathogen seems to employ membrane transporters and degradation enzymes to detoxify Eucalyptus phytoalexins. Remarkably, the Calonectria genome possesses a surprising number of secondary metabolism backbone enzyme genes involving toxin biosynthesis. It is also especially suited for cutin and lignin degradation. This indicates that toxin and cell wall degrading enzymes may act important roles in the establishment of Calonectria leaf blight. This study provides further understanding on the mechanism of pathogenesis in Calonectria.

Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass.

BACKGROUND: A number of Pyricularia species are known to infect different grass species. In the case of Pyricularia oryzae (syn. Magnaporthe oryzae), distinct populations are known to be adapted to a wide variety of grass hosts, including rice, wheat and many other grasses. The genome sizes of Pyricularia species are typical for filamentous ascomycete fungi [~ 40 Mbp for P. oryzae, and ~ 45 Mbp for P. grisea]. Genome plasticity, mediated in part by deletions promoted by recombination between repetitive elements [Genome Res 26:1091-1100, 2016, Nat Rev Microbiol 10:417-430,2012] and transposable elements [Annu Rev Phytopathol 55:483-503,2017] contributes to host adaptation. Therefore, comparisons of genome structure of individual species will provide insight into the evolution of host specificity. However, except for the P. oryzae subgroup, little is known about the gene content or genome organization of other Pyricularia species, such as those infecting Pennisetum grasses. RESULTS: Here, we report the genome sequence of P. penniseti strain P1609 isolated from a Pennisetum grass (JUJUNCAO) using PacBio SMRT sequencing technology. Phylogenomic analysis of 28 Magnaporthales species and 5 non-Magnaporthales species indicated that P1609 belongs to a Pyricularia subclade, which is genetically distant from P. oryzae. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires had diverged between P1609 and the P. oryzae strain 70-15, including the known avirulence genes, other putative secreted proteins, as well as some other predicted Pathogen-Host Interaction (PHI) genes. Genomic sequence comparison also identified many genomic rearrangements relative to P. oryzae. CONCLUSION: Our results suggested that the genomic sequence of the P. penniseti P1609 could be a useful resource for the genetic study of the Pennisetum-infecting Pyricularia species and provide new insight into evolution of pathogen genomes during host adaptation.

Methylmalonate-semialdehyde dehydrogenase mediated metabolite homeostasis essentially regulate conidiation, polarized germination and pathogenesis in Magnaporthe oryzae.

Plants generate multitude of aldehydes under abiotic and biotic stress conditions. Ample demonstrations have shown that rice-derived aldehydes enhance the resistance of rice against the rice-blast fungus Magnaporthe oryzae. However, how the fungal pathogen nullifies the inhibitory effects of host aldehydes to establish compatible interaction remains unknown. Here we identified and evaluated the in vivo transcriptional activities of M. oryzae aldehyde dehydrogenase (ALDH) genes. Transcriptional analysis of M. oryzae ALDH genes revealed that the acetylating enzyme Methylmalonate-Semialdehyde Dehydrogenase (MoMsdh/MoMmsdh) elevated activities during host invasion and colonization of the fungus. We further examined the pathophysiological importance of MoMSDH by deploying integrated functional genetics, and biochemical approaches. MoMSDH deletion mutant ΔMomsdh exhibited germination defect, hyper-branching of germ tube and failed to form appressoria on hydrophobic and hydrophilic surface. The MoMSDH disruption caused accumulation of small branch-chain amino acids, pyridoxine and AMP/cAMP in the ΔMomsdh mutant and altered Spitzenkörper organization in the conidia. We concluded that MoMSDH contribute significantly to the pathogenesis of M. oryzae by regulating the mobilization of Spitzenkörper during germ tube morphogenesis, appressoria formation by acting as metabolic switch regulating small branch-chain amino acids, inositol, pyridoxine and AMP/cAMP homeostasis.

Two different subcellular-localized Acetoacetyl-CoA acetyltransferases differentiate diverse functions in Magnaporthe oryzae.

The mevalonate pathway is an efficient biosynthesis pathway that yields isoprenoids for promoting different crucial cellular functions, including ergosterol synthesis and growth regulation. Acetoacetyl-CoA acetyltransferase (EC2.3.1.9) is the first major catalytic enzyme constituting the mevalonate pathway and catalyzes the transformation of Acetoacetyl-CoA from two molecules of acetyl-CoA enroute ergosterol production in fungi. We identified two homologous genes encoding Acetoacetyl-CoA acetyltransferase (MoAcat1 and MoAcat2) in Magnaporthe oryzae, the rice blast fungus. Phylogenetic analysis indicates these two genes have different evolutionary history. We subsequently, conducted targeted gene deletion using homologous recombination technology to ascertain the unique roles of the two MoAcat homologues during the fungal morphogenesis and pathogenesis. The findings from our investigations showed that the activity of MoAcat1 promoted virulence in the rice blast fungus as such, the ΔMoacat1 mutants generated exhibited defect in virulence, whilst ΔMoacat1 mutants did not portray growth defects. ΔMoacat2 mutants on the other hand were characterized by reduction in growth and virulence. Furthermore, MoAcat1 and MoAcat2 showed different expression patterns and subcellular localizations in M. oryzae. From our investigations we came to the conclusion that, different subcellular localization contributes to the diverse functions of MoAcat1 and MoAcat2, which helps the successful establishment of blast disease by promoting efficient development of cell morphology and effective colonization of host tissue.

Putative RhoGAP proteins orchestrate vegetative growth, conidiogenesis and pathogenicity of the rice blast fungus Magnaporthe oryzae.

Rho GTPases, acting as molecular switches, are involved in the regulation of diverse cellular functions. Rho GTPase activating proteins (Rho GAPs) function as negative regulators of Rho GTPases and are required for a variety of signaling processes in cell development. But the mechanisms underlying Rho GAPs in Rho-mediated signaling pathways in fungi are still elusive. There are eight RhoGAP domain-containing genes annotated in the Magnaporthe oryzae genome. To understand the function of these RhoGAP genes, we generated knockout mutants of each of the RhoGAP genes through a homologous recombination-based method. Phenotypic analysis showed that growth rate of aerial hyphae of the Molrg1 deletion mutant decreased dramatically. The ΔMolrg1 mutant showed significantly reduced conidiation and appressorium formation by germ tubes. Moreover, it lost pathogenicity completely. Deletion of another Rho GAP (MoRga1) resulted in high percentage of larger or gherkin-shaped conidia and slight decrease in conidiation. Appressorial formation of the ΔMoRga1 mutant was delayed significantly on hydrophobic surface, while the development of mycelial growth and pathogenicity in plants was not affected. Confocal fluorescence microscopy imaging showed that MoRga1-GFP localizes to septal pore of the conidium, and this localization pattern requires both LIM and RhoGAP domains. Furthermore, either deleting the LIM or RhoGAP domain or introducing an inactivating R1032A mutation in the RhoGAP domain of MoRga1 caused similar defects as the Morga1 deletion mutant in terms of conidial morphology and appressorial formation, suggesting that MoRga1 is a stage-specific regulator of conidial differentiation by regulating some specific Rho GTPases. In this regard, MoRga1 and MoLrg1 physically interacted with both MoRac1-CA and MoCdc42-CA in the yeast two-hybrid and pull-down assays, suggesting that the actions of these two GAPs are involved in MoRac1 and MoCdc42 pathways. On the other hand, six other putative Rho GAPs (MoRga2 to MoRga7) were dispensable for conidiation, vegetative growth, appressorial formation and pathogenicity, suggesting that these Rho GAPs function redundantly during fungal development. Taking together, Rho GAP genes play important roles in M. oryzae development and infectious processes through coordination and modulation of Rho GTPases.

H1 restricts euchromatin-associated methylation pathways from heterochromatic encroachment.

Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1 , suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.

H1 restricts euchromatin-associated methylation pathways from heterochromatic encroachment.

Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1, suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to the loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.

MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.

DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2 and MBD4 and show that MBD2 acts as a TE repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused activation at a small subset of TEs in the vegetative cell of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. TE activation in mbd2 became more significant in the mbd5 mbd6 and adcp1 mutant backgrounds, suggesting that MBD2 acts redundantly with other silencing pathways to repress TEs. Overall, our study identifies MBD2 as a methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

Intraspecific diploidization of a halophyte root fungus drives heterosis.

How organisms respond to environmental stress is a key topic in evolutionary biology. This study focused on the genomic evolution of Laburnicola rhizohalophila, a dark-septate endophytic fungus from roots of a halophyte. Chromosome-level assemblies were generated from five representative isolates from structured subpopulations. The data revealed significant genomic plasticity resulting from chromosomal polymorphisms created by fusion and fission events, known as dysploidy. Analyses of genomic features, phylogenomics, and macrosynteny have provided clear evidence for the origin of intraspecific diploid-like hybrids. Notably, one diploid phenotype stood out as an outlier and exhibited a conditional fitness advantage when exposed to a range of abiotic stresses compared with its parents. By comparing the gene expression patterns in each hybrid parent triad under the four growth conditions, the mechanisms underlying growth vigor were corroborated through an analysis of transgressively upregulated genes enriched in membrane glycerolipid biosynthesis and transmembrane transporter activity. In vitro assays suggested increased membrane integrity and lipid accumulation, as well as decreased malondialdehyde production under optimal salt conditions (0.3 M NaCl) in the hybrid. These attributes have been implicated in salinity tolerance. This study supports the notion that hybridization-induced genome doubling leads to the emergence of phenotypic innovations in an extremophilic endophyte.