Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.     

H3K36me2 methyltransferase NSD2 orchestrates epigenetic reprogramming during spermatogenesis

Spermatogenesis is precisely controlled by sophisticated gene expression programs and is driven by epigenetic reprogramming, including histone modification alterations and histone-to-protamine transition. Nuclear receptor binding SET domain protein 2 (Nsd2) is the predominant histone methyltransferase catalyzing H3K36me2 and its role in male germ cell development remains elusive. Here, we report that NSD2 protein is abundant in spermatogenic cells. Conditional loss of Nsd2 in postnatal germ cells impaired fertility owing to apoptosis of spermatocytes and aberrant spermiogenesis. Nsd2 deficiency results in dysregulation of thousands of genes and remarkable reduction of both H3K36me2 and H3K36me3 in spermatogenic cells, with H3K36me2 occupancy correlating positively with expression of germline genes. Nsd2 deficiency leads to H4K16ac elevation in spermatogenic cells, probably through interaction between NSD2 and PSMA8, which regulates acetylated histone degradation. We further reveal that Nsd2 deficiency impairs EP300-induced H4K5/8ac, recognized by BRDT to mediate the eviction of histones. Accordingly, histones are largely retained in Nsd2-deficient spermatozoa. In addition, Nsd2 deficiency enhances expression of protamine genes, leading to increased protamine proteins in Nsd2-deficient spermatozoa. Our findings thus reveal a previously unappreciated role of the Nsd2-dependent chromatin remodeling during spermatogenesis and provide clues to the molecular mechanisms in epigenetic abnormalities impacting male reproductive health.     

Histone H3 lysine 4 monomethylation (H3K4me1) and H3 lysine 9 monomethylation (H3K9me1): Distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells

Hyperglycemia/hyperinsulinemia are leading cause for the induction type 2 diabetes and the role of post-translational histone modifications in dysregulating the expression of genes has emerged as potential important contributor in the progression of disease. The paradoxical nature of histone H3-Lysine 4 and Lysine 9 mono-methylation (H3K4me1 and H3K9me1) in both gene activation and repression motivated us to elucidate the functional relationship of these histone modifications in regulating expression of genes under hyperglycaemic/hyperinsulinemic condition. Chromatin immunoprecipitation–microarray analysis (ChIP-chip) was performed with H3 acetylation, H3K4me1 and H3K9me1 antibody. CLUSTER analysis of ChIP-chip (Chromatin immunoprecipitation–microarray analysis) data showed that mRNA expression and H3 acetylation/H3K4me1 levels on genes were inversely correlated with H3K9me1 levels on the transcribed regions, after 30 min of insulin stimulation under hyperglycaemic condition. Interestingly, we provide first evidence regarding regulation of histone de/acetylases and de/methylases; Myst4, Jmjd2b, Aof1 and Set by H3Ac, H3K4me1 and H3K9me1 under hyperinsulinemic/hyperglycaemic condition. ChIP–qPCR analysis shows association of increased H3Ac/H3K4me1 and decreased levels of H3K9me1 in up regulation of Myst4, Jmjd2, Set and Aof1 genes. We further analyse promoter occupancy of histone modifications by ChIP walking and observed increased occupancy of H3Ac/H3K4me1 on promoter region (−1000 to −1) of active genes and H3K9me1 on inactive genes under hyperglycemic/hyperinsulinemic condition. To best of our knowledge this is the first report that shows regulation of chromatin remodelling genes by alteration in the occupancy of histone H3Ac/H3K4/K9me on both promoter and transcribed regions.     

To incise or not and where: SET-domain methyltransferases know

The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3–9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.     

G9α‐dependent histone H3K9me3 hypomethylation promotes overexpression of cardiomyogenesis‐related genes in foetal mice

Alcohol consumption during pregnancy can cause foetal alcohol syndrome and congenital heart disease. Nonetheless, the underlying mechanism of alcohol‐induced cardiac dysplasia remains unknown. We previously reported that alcohol exposure during pregnancy can cause abnormal expression of cardiomyogenesis‐related genes, and histone H3K9me3 hypomethylation was observed in alcohol‐treated foetal mouse heart. Hence, an imbalance in histone methylation may be involved in alcohol‐induced cardiac dysplasia. In this study, we investigated the involvement of G9α histone methyltransferase in alcohol‐induced cardiac dysplasia in vivo and in vitro using heart tissues of foetal mice and primary cardiomyocytes of neonatal mice. Western blotting revealed that alcohol caused histone H3K9me3 hypomethylation by altering G9α histone methyltransferase expression in cardiomyocytes. Moreover, overexpression of cardiomyogenesis‐related genes (MEF2C, Cx43, ANP and β‐MHC) was observed in alcohol‐exposed foetal mouse heart. Additionally, we demonstrated that G9α histone methyltransferase directly interacted with histone H3K9me3 and altered its methylation. Notably, alcohol did not down‐regulate H3K9me3 methylation after G9α suppression by short hairpin RNA in primary mouse cardiomyocytes, preventing MEF2C, Cx43, ANP and β‐MHC overexpression. These findings suggest that G9α histone methyltransferase‐mediated imbalance in histone H3K9me3 methylation plays a critical role in alcohol‐induced abnormal expression cardiomyogenesis‐related genes during pregnancy. Therefore, G9α histone methyltransferase may be an intervention target for congenital heart disease.     

Regulation of H3K27me3 and H3K4me3 during early porcine embryonic development

The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co‐factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre‐implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1‐ to the 4‐cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1‐cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4‐cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4‐cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst. Mol. Reprod. Dev. 77: 540–549, 2010. © 2010 Wiley‐Liss, Inc.     

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.     

Jumonji C Domain Protein JMJ705-Mediated Removal of Histone H3 Lysine 27 Trimethylation Is Involved in Defense-Related Gene Activation in Rice

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate–induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.     

The trxG family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine‐tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 (SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late‐flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 (atx1), sdg25 and curly leaf (clf), as well as the demethylase double mutant lsd1‐like1 lsd1‐like2 (ldl1 ldl2). We found that the early‐flowering mutants sdg25, atx1 and clf interact antagonistically with the late‐flowering mutant sdg26, whereas the late‐flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/AGAMOUS‐LIKE 20 (SOC1/AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late‐flowering phenotype.     

The Phytophthora effector Avh241 interacts with host NDR1-like proteins to manipulate plant immunity

Plant pathogens rely on effector proteins to suppress host innate immune responses and facilitate colonization. Although the Phytophthora sojae RxLR effector Avh241 promotes Phytophthora infection, the molecular basis of Avh241 virulence remains poorly understood. Here we identified non-race specific disease resistance 1 (NDR1)-like proteins, the critical components in plant effector-triggered immunity (ETI) responses, as host targets of Avh241. Avh241 interacts with NDR1 in the plasma membrane and suppresses NDR1-participated ETI responses. Silencing of GmNDR1s increases the susceptibility of soybean to P. sojae infection, and overexpression of GmNDR1s reduces infection, which supports its positive role in plant immunity against P. sojae. Furthermore, we demonstrate that GmNDR1 interacts with itself, and Avh241 probably disrupts the self-association of GmNDR1. These data highlight an effective counter-defense mechanism by which a Phytophthora effector suppresses plant immune responses, likely by disturbing the function of NDR1 during infection.     

Narrow H3K4me2 is required for chicken PGC formation

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC‐related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor‐β). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.