Ustilaginoidea virens-secreted effector Uv1809 suppresses rice immunity by enhancing OsSRT2-mediated histone deacetylation.

Rice false smut caused by Ustilaginoidea virens is a devastating rice (Oryza sativa) disease worldwide. However, the molecular mechanisms underlying U. virens-rice interactions are largely unknown. In this study, we identified a secreted protein, Uv1809, as a key virulence factor. Heterologous expression of Uv1809 in rice enhanced susceptibility to rice false smut and bacterial blight. Host-induced gene silencing of Uv1809 in rice enhanced resistance to U. virens, suggesting that Uv1809 inhibits rice immunity and promotes infection by U. virens. Uv1809 suppresses rice immunity by targeting and enhancing rice histone deacetylase OsSRT2-mediated histone deacetylation, thereby reducing H4K5ac and H4K8ac levels and interfering with the transcriptional activation of defence genes. CRISPR-Cas9 edited ossrt2 mutants showed no adverse effects in terms of growth and yield but displayed broad-spectrum resistance to rice pathogens, revealing a potentially valuable genetic resource for breeding disease resistance. Our study provides insight into defence mechanisms against plant pathogens that inactivate plant immunity at the epigenetic level.

Post-Translational Modification ß-Hydroxybutyrylation Regulates Ustilaginoidea virens Virulence.

Lysine ß-hydroxybutyrylation (Kbhb) is an evolutionarily conserved and widespread post-translational modification that is associated with active gene transcription and cellular proliferation. However, its role in phytopathogenic fungi remains unknown. Here, we characterized Kbhb in the rice false smut fungus Ustilaginoidea virens. We identified 2204 Kbhb sites in 852 proteins, which are involved in diverse biological processes. The mitogen-activated protein kinase UvSlt2 is a Kbhb protein, and a strain harboring a point mutation at K72, the Kbhb site of this protein, had decreased UvSlt2 activity and reduced fungal virulence. Molecular dynamic simulations revealed that K72bhb increases the hydrophobic solvent-accessible surface area of UvSlt2, thereby affecting its binding to its substrates. The mutation of K298bhb in the septin UvCdc10 resulted in reduced virulence and altered the subcellular localization of this protein. Moreover, we confirmed that the NAD+-dependent histone deacetylases UvSirt2 and UvSirt5 are the major enzymes that remove Kbhb in U. virens. Collectively, our findings identify regulatory elements of the Kbhb pathway and reveal important roles for Kbhb in regulating protein localization and enzymatic activity. These findings provide insight into the regulation of virulence in phytopathogenic fungi via post-translational modifications.

ACL and HAT1 form a nuclear module to acetylate histone H4K5 and promote cell proliferation.

Acetyl-CoA utilized by histone acetyltransferases (HAT) for chromatin modification is mainly generated by ATP-citrate lyase (ACL) from glucose sources. How ACL locally establishes acetyl-CoA production for histone acetylation remains unclear. Here we show that ACL subunit A2 (ACLA2) is present in nuclear condensates, is required for nuclear acetyl-CoA accumulation and acetylation of specific histone lysine residues, and interacts with Histone AcetylTransferase1 (HAT1) in rice. The rice HAT1 acetylates histone H4K5 and H4K16 and its activity on H4K5 requires ACLA2. Mutations of rice ACLA2 and HAT1 (HAG704) genes impair cell division in developing endosperm, result in decreases of H4K5 acetylation at largely the same genomic regions, affect the expression of similar sets of genes, and lead to cell cycle S phase stagnation in the endosperm dividing nuclei. These results indicate that the HAT1-ACLA2 module selectively promotes histone lysine acetylation in specific genomic regions and unravel a mechanism of local acetyl-CoA production which couples energy metabolism with cell division.

The opening of mitochondrial permeability transition pore (mPTP) and the inhibition of electron transfer chain (ETC) induce mitophagy in wheat roots under waterlogging stress.

Mitochondria are crucial for the regulation of intracellular energy metabolism, biosynthesis, and cell survival. And studies have demonstrated the role of mitochondria in oxidative stress-induced autophagy in plants. Previous studies found that waterlogging stress can induce the opening of mitochondrial permeability transition pore (mPTP) and the release of cytochrome c in endosperm cells, which proved that mPTP plays an important role in the programmed cell death of endosperm cells under waterlogging stress. This study investigated the effects of the opening of mPTP and the inhibition of ETC on mitophagy in wheat roots under waterlogging stress. The results showed that autophagy related genes in the mitochondria of wheat root cells could respond to waterlogging stress; waterlogging stress led to the degradation of the characteristic proteins cytochrome c and COXII in the mitochondria of root cells. With the prolongation of waterlogging time, the protein degradation degree and the occurrence of mitophagy gradually increased. Under waterlogging stress, exogenous mPTP opening inhibitor CsA inhibited mitophagy in root cells and alleviated mitophagy induced by flooding stress, while exogenous mPTP opening inducer CCCP induced mitophagy in root cells; exogenous mPTP opening inducer CCCP induced mitophagy in root cells. The electron transfer chain inhibitor antimycin A induces mitophagy in wheat root cells and exacerbates mitochondrial degradation. In conclusion, waterlogging stress led to the degradation of mitochondrial characteristic proteins and the occurrence of mitophagy in wheat root cells, and the opening of mPTP and the inhibition of ETC induced the occurrence of mitophagy.

Comparative oxidation proteomics analyses suggest redox regulation of cytosolic translation in rice leaves upon Magnaporthe oryzae infection.

Pathogen attack can increase plant levels of reactive oxygen species (ROS), which act as signaling molecules to activate plant defense mechanisms. Elucidating these processes is crucial for understanding redox signaling pathways in plant defense responses. Using an iodo-tandem mass tag (TMT)-based quantitative proteomics approach, we mapped 3362 oxidized cysteine sites in 2275 proteins in rice leaves. Oxidized proteins were involved in gene expression, peptide biosynthetic processes, stress responses, ROS metabolic processes, and translation pathways. Magnaporthe oryzae infection led to increased oxidative modification levels of 512 cysteine sites in 438 proteins, including many transcriptional regulators and ribosomal proteins. Ribosome profiling (Ribo-seq) analysis revealed that the oxidative modification of ribosomal proteins promoted the translational efficiency of many mRNAs involved in defense response pathways, thereby affecting rice immunity. Our results suggest that increased oxidative modification of ribosomal proteins in rice leaves promotes cytosolic translation, thus revealing a novel function of post-translational modifications. Furthermore, the oxidation-sensitive proteins identified here provide a valuable resource for research on protein redox regulation and can guide future mechanistic studies.

ROS-stimulated protein lysine acetylation is required for crown root development in rice.

INTRODUCTION: As signal molecules in aerobic organisms, locally accumulated ROS have been reported to balance cell division and differentiation in the root meristem. Protein posttranslational modifications such as lysine acetylation play critical roles in controlling a variety of cellular processes. However, the mechanism by which ROS regulate root development is unknown. In addition, how protein lysine acetylation is regulated and whether cellular ROS levels affect protein lysine acetylation remain unclear. OBJECTIVES: We aimed to elucidate the relationship between ROS and protein acetylation by exploring a rice mutant plant that displays a decreased level of ROS in postembryonic crown root (CR) cells and severe defects in CR development. METHODS: First, proteomic analysis was used to find candidate proteins responsible for the decrease of ROS detected in the wox11 mutant. Then, biochemical, molecular, and genetic analyses were used to study WOX11-regulated genes involved in ROS homeostasis. Finally, acetylproteomic analysis of wild type and wox11 roots treated with or without potassium iodide (KI) and hydrogen peroxide (H2O2) was used to study the effects of ROS on protein acetylation in rice CR cells. RESULTS: We demonstrated that WOX11 was required to maintain ROS homeostasis by upregulating peroxidase genes in the crown root meristem. Acetylproteomic analysis revealed that WOX11-dependent hydrogen peroxide (H2O2) levels in CR cells promoted lysine acetylation of many non-histone proteins enriched for nitrogen metabolism and peptide/protein synthesis pathways. Further analysis revealed that the redox state affected histone deacetylases (HDACs) activity, which was likely related to the high levels of protein lysine acetylation in CR cells. CONCLUSION: WOX11-controlled ROS level in CR meristem cells is required for protein lysine acetylation which represents a mechanism of ROS-promoted CR development in rice.

Ustilaginoidea virens modulates lysine 2-hydroxyisobutyrylation in rice flowers during infection.

The post-translational modification lysine 2-hydroxyisobutyrylation (Khib ) plays an important role in gene transcription, metabolism, and enzymatic activity. Khib sites have been identified in rice (Oryza sativa). However, the Khib status of proteins in rice flowers during pathogen infection remains unclear. Here, we report a comprehensive identification of Khib -modified proteins in rice flowers, and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens. By using a tandem mass tag-based quantitative proteomics approach, we identified 2,891 Khib sites on 964 proteins in rice flowers. Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Khib levels were substantially reduced upon infection with U. virens. Chromatin immunoprecipitation polymerase chain reaction (PCR) and reverse transcription quantitative PCR analyses revealed that histone Khib is involved in the expression of disease-resistance genes. More importantly, most quantified sites on core histones H3 were downregulated upon U. virens infection. In addition, the histone deacetylases HDA705, HDA716, SRT1, and SRT2 are involved in the removal of Khib marks in rice. HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U. virens, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae (Xoo). Our data suggest that U. virens could modulate Khib in rice flowers during infection.

Histone deacetylases control lysine acetylation of ribosomal proteins in rice.

Lysine acetylation (Kac) is well known to occur in histones for chromatin function and epigenetic regulation. In addition to histones, Kac is also detected in a large number of proteins with diverse biological functions. However, Kac function and regulatory mechanism for most proteins are unclear. In this work, we studied mutation effects of rice genes encoding cytoplasm-localized histone deacetylases (HDAC) on protein acetylome and found that the HDAC protein HDA714 was a major deacetylase of the rice non-histone proteins including many ribosomal proteins (r-proteins) and translation factors that were extensively acetylated. HDA714 loss-of-function mutations increased Kac levels but reduced abundance of r-proteins. In vitro and in vivo experiments showed that HDA714 interacted with r-proteins and reduced their Kac. Substitutions of lysine by arginine (depleting Kac) in several r-proteins enhance, while mutations of lysine to glutamine (mimicking Kac) decrease their stability in transient expression system. Ribo-seq analysis revealed that the hda714 mutations resulted in increased ribosome stalling frequency. Collectively, the results uncover Kac as a functional posttranslational modification of r-proteins which is controlled by histone deacetylases, extending the role of Kac in gene expression to protein translational regulation.

Short-term waterlogging-induced autophagy in root cells of wheat can inhibit programmed cell death.

Autophagy is a pathway for the degradation of cytoplasmic components in eukaryotes. In wheat, the mechanism by which autophagy regulates programmed cell death (PCD) is unknown. Here, we demonstrated that short-term waterlogging-induced autophagy inhibited PCD in root cells of wheat. The waterlogging-tolerant wheat cultivar Huamai 8 and the waterlogging-sensitive wheat cultivar Huamai 9 were used as experimental materials, and their roots were waterlogged for 0-48 h. Waterlogging stress increased the number of autophagic structures, the expression levels of autophagy-related genes (TaATG), and the occurrence of PCD in root cells. PCD manifested as morphological changes in the cell nucleus, significant enhancement of DNA laddering bands, and increases in caspase-like protease activity and the expression levels of metacaspase genes. The autophagy promoter rapamycin (RAPA) reduced PCD levels, whereas the autophagy inhibitor 3-methyladenine (3-MA) enhanced them. The expression levels of TaATG genes and the number of autophagic structures were lower in cortex cells than in stele cells, but the levels of PCD were higher in cortex cells. The number of autophagic structures was greater in Huamai 8 than in Huamai 9, but the levels of PCD were lower. In summary, our results showed that short-term waterlogging induced autophagy which could inhibit PCD. Mechanisms of response to waterlogging stress differed between cortex and stele cells and between two wheat cultivars of contrasting waterlogging tolerance.

Mutual regulation of ROS accumulation and cell autophagy in wheat roots under hypoxia stress.

Here, we explored the mutual regulation of radical oxygen species (ROS) and autophagy in wheat (Triticum aestivum L.) roots under hypoxia stress. We also analyzed differences between the responses of the stele and the cortex in the two wheat cultivars Huamai 8 (waterlogging-tolerant) and Huamai 9 (waterlogging-sensitive) to hypoxia stress. In situ detection and ultracytochemical localization analysis in wheat roots showed that hypoxia stress caused greater increases in ROS levels and the expression levels of alternative oxidase (AOX) and antioxidant enzymes in the stele than in the cortex. The analysis of exogenous ROS addition and the inhibition of its production revealed the pivotal roles played by ROS in autophagy. Moreover, transmission electron microscopy and qRT-PCR analysis indicated that the stele had a higher level of autophagy than the cortex and that the two wheat cultivars primarily differed in the type and number of autophagosomes. Additional research revealed that autophagy could remove excess ROS, as pre-treatment with the autophagy inhibitor 3-methyladenine increased ROS levels in roots and the addition of the autophagy inducer rapamycin reduced root ROS levels. In conclusion, hypoxia stress induced ROS accumulation in wheat roots where ROS acted as an autophagy signal. Furthermore, higher levels of autophagy and antioxidant enzyme expression in the stele facilitated the elimination of oxidative damage caused by excessive ROS and thereby increased cell survival; in the cortex, a large number of cells died and formed aerenchyma.

Histone deacetylase HDA710 controls salt tolerance by regulating ABA signaling in rice.

Plants have evolved numerous mechanisms that assist them in withstanding environmental stresses. Histone deacetylases (HDACs) play crucial roles in plant stress responses; however, their regulatory mechanisms remain poorly understood. Here, we explored the function of HDA710/OsHDAC2, a member of the HDAC RPD3/HDA1 family, in stress tolerance in rice (Oryza sativa). We established that HDA710 localizes to both the nucleus and cytoplasm and is involved in regulating the acetylation of histone H3 and H4, specifically targeting H4K5 and H4K16 under normal conditions. HDA710 transcript accumulation levels were strongly induced by abiotic stresses including drought and salinity, as well as by the phytohormones jasmonic acid (JA) and abscisic acid (ABA). hda710 knockout mutant plants showed enhanced salinity tolerance and reduced ABA sensitivity, whereas transgenic plants overexpressing HDA710 displayed the opposite phenotypes. Moreover, ABA- and salt-stress-responsive genes, such as OsLEA3, OsABI5, OsbZIP72, and OsNHX1, were upregulated in hda710 compared with wild-type plants. These expression differences corresponded with higher levels of histone H4 acetylation in gene promoter regions in hda710 compared with the wild type under ABA and salt-stress treatment. Collectively, these results suggest that HDA710 is involved in regulating ABA- and salt-stress-responsive genes by altering H4 acetylation levels in their promoters. This article is protected by copyright. All rights reserved.

Dynamics and functional interplay of histone lysine butyrylation, crotonylation, and acetylation in rice under starvation and submergence.

BACKGROUND: Histone lysine acylations by short-chain fatty acids are distinct from the widely studied histone lysine acetylation in chromatin, although both modifications are regulated by primary metabolism in mammalian cells. It remains unknown whether and how histone acylation and acetylation interact to regulate gene expression in plants that have distinct regulatory pathways of primary metabolism. RESULTS: We identify 4 lysine butyrylation (Kbu) sites (H3K14, H4K12, H2BK42, and H2BK134) and 45 crotonylation (Kcr) sites on rice histones by mass spectrometry. Comparative analysis of genome-wide Kbu and Kcr and H3K9ac in combination with RNA sequencing reveals 25,306 genes marked by Kbu and Kcr in rice and more than 95% of H3K9ac-marked genes are marked by both. Kbu and Kcr are enriched at the 5' region of expressed genes. In rice under starvation and submergence, Kbu and Kcr appear to be less dynamic and display changes in different sets of genes compared to H3K9ac. Furthermore, Kbu seems to preferentially poise gene activation by external stresses, rather than internal circadian rhythm which has been shown to be tightly associated with H3K9ac. In addition, we show that rice sirtuin histone deacetylase (SRT2) is involved in the removal of Kcr. CONCLUSION: Kbu, Kcr, and H3K9ac redundantly mark a large number of active genes but display different responses to external and internal signals. Thus, the proportion of rice histone lysine acetylation and acylation is dynamically regulated by environmental and metabolic cues, which may represent an epigenetic mechanism to fine-tune gene expression for plant adaptation.

Reactive oxygen species regulate programmed cell death progress of endosperm in winter wheat (Triticum aestivum L.) under waterlogging.

Previous studies have proved that waterlogging stress accelerates the programmed cell death (PCD) progress of wheat endosperm cells. A highly waterlogging-tolerant wheat cultivar Hua 8 and a waterlogging susceptible wheat cultivar Hua 9 were treated with different waterlogging durations, and then, dynamic changes of reactive oxygen species (ROS), gene expressions, and activities of antioxidant enzymes in endosperm cells were detected. The accumulation of ROS increased considerably after 7 days of waterlogging treatment (7 DWT) and 12 DWT in both cultivars compared with control group (under non-waterlogged conditions), culminated at 12 DAF (days after flowering) and reduced hereafter. Waterlogging resulted in a great increase of H2O2 and O2 (-) in plasma membranes, cell walls, mitochondrias, and intercellular spaces with ultracytochemical localization. Moreover, the deformation and rupture of cytomembranes as well as the swelling and distortion of mitochondria were obvious. Under waterlogging treatment conditions, catalase (CAT) gene expression increased in endosperm of Hua 8 but activity decreased. In addition, Mn superoxide dismutase (MnSOD) gene expression and superoxide dismutase (SOD) activity increased. Compared with Hua 8, both CAT, MnSOD gene expressions and CAT, SOD activities decreased in Hua 9. Moreover, ascorbic acid and mannitol relieve the intensifying of PCD processes in Hua 8 endosperm cells induced by waterlogging. These results indicate that ROS have important roles in the PCD of endosperm cells, the changes both CAT, MnSOD gene expressions and CAT, SOD activities directly affected the accumulation of ROS in two different wheat cultivars under waterlogging, ultimately led to the PCD acceleration of endosperm.

Regulation of histone methylation and reprogramming of gene expression in the rice inflorescence meristem.

Rice inflorescence meristem (IM) activity is essential for panicle development and grain production. How chromatin and epigenetic mechanisms regulate IM activity remains unclear. Genome-wide analysis revealed that in addition to genes involved in the vegetative to reproductive transition, many metabolic and protein synthetic genes were activated in IM compared with shoot apical meristem and that a change in the H3K27me3/H3K4me3 ratio was an important factor for the differential expression of many genes. Thousands of genes gained or lost H3K27me3 in IM, and downregulation of the H3K27 methyltransferase gene SET DOMAIN GROUP 711 (SDG711) or mutation of the H3K4 demethylase gene JMJ703 eliminated the increase of H3K27me3 in many genes. SDG711-mediated H3K27me3 repressed several important genes involved in IM activity and many genes that are silent in the IM but activated during floral organogenesis or other developmental stages. SDG711 overexpression augmented IM activity and increased panicle size; suppression of SDG711 by RNA interference had the opposite effect. Double knockdown/knockout of SDG711 and JMJ703 further reduced panicle size. These results suggest that SDG711 and JMJ703 have agonistic functions in reprogramming the H3K27me3/H3K4me3 ratio and modulating gene expression in the IM.