Plant histone variants at the nexus of chromatin readouts, stress and development.

Histones are crucial proteins that are involved in packaging the DNA as condensed chromatin inside the eukaryotic cell nucleus. Rather than being static packaging units, these molecules undergo drastic variations spatially and temporally to facilitate accessibility of DNA to replication, transcription as well as wide range of gene regulatory machineries. In addition, incorporation of paralogous variants of canonical histones in the chromatin is ascribed to specific functions. Given the peculiar requirement of plants to rapidly modulate gene expression levels on account of their sessile nature, histones and their variants serve as additional layers of gene regulation. This review summarizes the mechanisms and implications of distribution, modifications and differential incorporation of histones and their variants across plant genomes, and outlines emerging themes.

Novel regulators of mitochondrial genome integrity.

Mitochondrial genomes are unusual in that they have very low mutation rates in coding sequences when compared with animals, yeast, and bacteria. It has been proposed that the mitochondrial (mt) DNA damage is preferentially repaired by homologous recombination (HR). In this short review, we summarize and discuss the well-established and recently identified pathways that help in maintaining the integrity of plant mitochondrial genomes.

Plant polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains.

Across eukaryotes, gene regulation is manifested via chromatin states roughly distinguished as heterochromatin and euchromatin. The establishment, maintenance, and modulation of the chromatin states is mediated using several factors including chromatin modifiers. However, factors that avoid the intrusion of silencing signals into protein-coding genes are poorly understood. Here we show that a plant specific paralog of RNA polymerase (Pol) II, named Pol IV, is involved in avoidance of facultative heterochromatic marks in protein-coding genes, in addition to its well-established functions in silencing repeats and transposons. In its absence, H3K27 trimethylation (me3) mark intruded the protein-coding genes, more profoundly in genes embedded with repeats. In a subset of genes, spurious transcriptional activity resulted in small(s) RNA production, leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with a larger genome with distributed heterochromatin compared with Arabidopsis Our results indicate the division of labor among plant-specific polymerases, not just in establishing effective silencing via sRNAs and DNA methylation but also in influencing chromatin boundaries.

Investigation of Transposon DNA Methylation and Copy Number Variation in Plants Using Southern Hybridisation.

Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops. Here, we present a simple reproducible Southern hybridisation-based assay to obtain incontrovertible methylation patterns from targeted regions in the rice genome. By employing minor but key modifications, we reliably detected transposon copy number variations over multiple generations. This method can be regarded as a gold standard for validation of epigenetic variations at target loci, and the consequent proliferation of transposons, or segregation in several plant replicates and genotypes. Graphical abstract.

Impact of Phyllosphere Methylobacterium on Host Rice Landraces.

The genus Methylobacterium includes widespread plant-associated bacteria that are abundant in the plant phyllosphere (leaf surfaces), consume plant-secreted methanol, and can produce plant growth-promoting metabolites. However, despite the potential to increase agricultural productivity, their impact on host fitness in the natural environment is relatively poorly understood. Here, we conducted field experiments with three traditionally cultivated rice landraces from northeastern India. We inoculated seedlings with native versus nonnative phyllosphere Methylobacterium strains and found significant impacts on plant growth and grain yield. However, these effects were variable. Whereas some Methylobacterium isolates were beneficial for their host, others had no impact or were no more beneficial than the bacterial growth medium on its own. Host plant benefits were not consistently associated with Methylobacterium colonization and did not have altered phyllosphere microbiome composition, changes in the early expression of plant stress response pathways, or bacterial auxin production. We provide the first demonstration of the benefits of phyllosphere Methylobacterium for rice yield under field conditions and highlight the need for further analysis to understand the mechanisms underlying these benefits. Given that the host landrace-Methylobacterium relationship was not generalizable, future agricultural applications will require careful testing to identify coevolved host-bacterium pairs that may enhance the productivity of high-value rice varieties. IMPORTANCE Plants are associated with diverse microbes in nature. Do the microbes increase host plant health, and can they be used for agricultural applications? This is an important question that must be answered in the field rather than in the laboratory or greenhouse. We tested the effects of native, leaf-inhabiting bacteria (genus Methylobacterium) on traditionally cultivated rice varieties in a crop field. We found that inoculation with some bacteria increased rice grain production substantially while a nonnative bacterium reduced plant health. Overall, the effect of bacterial inoculation varied across pairs of rice varieties and their native bacteria. Thus, knowledge of evolved associations between specific bacteria hosted by specific rice varieties is necessary to develop ways to increase the yield of traditional rice landraces and preserve these important sources of cultural and genetic diversity.

Cultivar-specific miRNA-mediated RNA silencing in grapes.

MAIN CONCLUSION: In-depth comparative degradome analysis of two domesticated grape cultivars with diverse secondary metabolite accumulation reveals differential miRNA-mediated targeting. Small (s)RNAs such as micro(mi)RNAs and secondary small interfering (si) often work as negative switches of gene expression. In plants, it is well known that miRNAs target and cleave mRNAs that have high sequence complementarity. However, it is not known if there are variations in miRNA-mediated targeting between subspecies and cultivars that have been subjected to vast genetic modifications through breeding and other selections. Here, we have used PAREsnip2 tool for analysis of degradome datasets derived from two contrasting domesticated grape cultivars having varied fruit color, habit and leaf shape. We identified several interesting variations in sRNA targeting using degradome and 5'RACE analysis between two contrasting grape cultivars that was further correlated using RNA-seq analysis. Several of the differences we identified are associated with secondary metabolic pathways. We propose possible means by which sRNAs might contribute to diversity in secondary metabolites and other development pathways between two domesticated cultivars of grapes.

Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs.

BACKGROUND: Small non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets. RESULTS: Here, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets. CONCLUSIONS: We identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

Plant-virus-insect tritrophic interactions: insights into the functions of geminivirus virion-sense strand genes.

The genome of the plant-infecting viruses in the family Geminiviridae is composed of one or two circular single stranded DNA of approximately 2.7-5.2 kb in length. These viruses have emerged as the most devastating pathogen infecting a large number of crops and weeds across the continents. They code for fewer open reading frames (ORFs) through the generation of overlapping transcripts derived from the bidirectional viral promoters. Members of geminiviruses code for up to four ORFs in the virion-sense strand, and their gene expression is regulated by various cis-elements located at their promoters in the intergenic region. These viral proteins perform multiple functions at every stage of the viral life cycle such as virus transport, insect-mediated virus transmission and suppression of host defence. They impede the host's multi-layered antiviral mechanisms including gene silencing (at transcriptional and post-transcriptional levels) and hypersensitive response. This review summarizes the essential role of virion-sense strand encoded proteins in transport of viral genomes within and between plant cells, countering defence in hosts (both plants and the insects), and also in the ubiquitous role in vector-mediated transmission. We highlight the significance of their pro-viral activities in manipulating host-derived innate immune responses and the interaction with whitefly-derived proteins. We also discuss the current knowledge on virus replication and transcription within the insect body.

Stability of Begomoviral pathogenicity determinant ßC1 is modulated by mutually antagonistic SUMOylation and SIM interactions.

BACKGROUND: To successfully invade new hosts, plant viruses must break host resistance and be competent to move within and between plant cells. As a means, viral proteins known as pathogenicity determinants have evolved to coordinate a network of protein interactions. The ßC1 protein encoded by specific geminiviral satellites acts as a key pathogenicity determinant for this disease-causing family of plant viruses. Post-translational modifications (PTMs) such as ubiquitination and phosphorylation of the ßC1 protein have been shown to occur in diverse viruses. However, the relevance of these and other layers of PTMs in host-geminiviral interactions has not been fully understood. RESULTS: Here we identified the significance of a novel layer of PTMs in the ßC1 protein of Synedrella yellow vein clearing virus (SyYVCV), a newly identified member of the Begomovirus genus of Geminiviruses. This protein has conserved SUMOylation and SUMO-interacting motifs (SIMs), and we observed SUMOylation of SyYVCV ßC1 in host plants as a defensive strategy against ubiquitin-mediated degradation. Counteracting this, SIMs encoded in ßC1 mediate the degradation of ßC1; however, both these PTMs are essential for the function of ßC1 protein since SIM and SUMOylation motif mutants failed to promote pathogenicity and viral replication in vivo. SUMOylation in different motifs of ßC1 led to functionally distinct outcomes, regulating the stability and function of the ßC1 protein, as well as increased global SUMOylation of host proteins. CONCLUSION: Our results indicate the presence of a novel mechanism mediating a fine balance between defence and counter-defence in which a SIM site is competitively sought for degradation and, as a counter-defence, ßC1 undergoes SUMOylation to escape from its degradation.

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs.

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

Loss of function of Oryza sativa Argonaute 18 induces male sterility and reduction in phased small RNAs.

KEY MESSAGE: In this manuscript, we show that Oryza sativa indica Argonaute protein AGO18 is required for male gametophyte development likely to through a small RNA-mediated mechanism. Monocots have evolved unique gene silencing pathways due to the presence of unique members of Dicer-like and Argonaute (AGO) family members. Among the monocot AGO homologs, AGO18 occupies a unique position. Previous reports have implicated this protein in viral resistance as well as in gametogenesis, likely through its competition with AGO1 clade members for micro(mi)RNAs and other small (s)RNAs. Although expression of rice AGO18 in specific stages of male gametogenesis has been documented, its major functions in plant development remain poorly understood. Here, we show that Oryza sativa indica AGO18 is involved in male gametophyte development. Knockdown (KD) of AGO18 in transgenic rice lines resulted in stunted plants that are male sterile, whereas their carpels were functional. Transcriptome analysis revealed downregulation of several pollen development-associated genes in KD lines. sRNA sequencing in vegetative and reproductive tissues of KD lines indicated reduction of miRNAs and phased secondary sRNAs implicated in male gametophyte development. Our results indicate a distinct role for rice AGO18 in male fertility.

Phenotypic diversity of Methylobacterium associated with rice landraces in North-East India.

The ecology and distribution of many bacteria is strongly associated with specific eukaryotic hosts. However, the impact of such host association on bacterial ecology and evolution is not well understood. Bacteria from the genus Methylobacterium consume plant-derived methanol, and are some of the most abundant and widespread plant-associated bacteria. In addition, many of these species impact plant fitness. To determine the ecology and distribution of Methylobacterium in nature, we sampled bacteria from 36 distinct rice landraces, traditionally grown in geographically isolated locations in North-East (NE) India. These landraces have been selected for diverse phenotypic traits by local communities, and we expected that the divergent selection on hosts may have also generated divergence in associated Methylobacterium strains. We determined the ability of 91 distinct rice-associated Methylobacterium isolates to use a panel of carbon sources, finding substantial variability in carbon use profiles. Consistent with our expectation, across spatial scales this phenotypic variation was largely explained by host landrace identity rather than geographical factors or bacterial taxonomy. However, variation in carbon utilisation was not correlated with sugar exudates on leaf surfaces, suggesting that bacterial carbon use profiles do not directly determine bacterial colonization across landraces. Finally, experiments showed that at least some rice landraces gain an early growth advantage from their specific phyllosphere-colonizing Methylobacterium strains. Together, our results suggest that landrace-specific host-microbial relationships may contribute to spatial structure in rice-associated Methylobacterium in a natural ecosystem. In turn, association with specific bacteria may provide new ways to preserve and understand diversity in one of the most important food crops of the world.

Extraction and Purification of Laccases from Rice Stems.

Laccases are found in cell walls of plants in very low amounts. This protocol provides an efficient method to purify laccases from rice stems. The method involves three steps: 1) Isolation of total protein from rice stems using buffers with high salt concentration to extract protein from cell walls; 2) Purification of laccases using concanavalin-A beads; and, 3) In-gel staining of laccases with 4-hydroxyindole. Concanavalin-A specifically binds to internal or non-reducing terminal α-D-mannosyl and α-D-glucosyl groups found in glycoproteins and glycolipids. Laccases being glycoproteins binds to concanavalin-A during purification process and eluted with mannose.

Major Domestication-Related Phenotypes in Indica Rice Are Due to Loss of miRNA-Mediated Laccase Silencing.

Domestication of rice (Oryza sativa) included conversion of perennial wild species with few seeds to short plants that produced abundant seeds. Most domestication-associated changes were due to variations in transcription factors and other key proteins such as enzymes. Here, we show that multiple yield-related traits associated with indica rice domestication are linked to micro (mi) RNA-mediated regulation. Analysis of small (s) RNA data sets from cultivated indica rice lines, a few landraces, and two wild relatives of rice revealed the presence of abundant 22-nucleotide (nt) reads in wild relatives that mapped to miR397 precursors. miR397 was expressed at very high levels in wild relatives and at negligible levels in high-yielding cultivated lines. In its genera-specific form of 22-nt, miR397 targeted mRNAs encoding laccases that decayed and induced robust secondary cascade silencing in wild species that required RNA-dependent RNA polymerase 6. In wild species of rice, reduced expression of laccases resulted in low lignification. As expected, overexpression of miR397 induced de-domestication phenotypes. At least 26 uncharacterized QTLs previously implicated in rice yield overlapped with laccases and miR397 genes. These results suggest that miRNAs contribute to rice domestication-associated phenotypes.

Molecular characterization of a new begomovirus infecting Synedrella nodiflora in South India.

Begomoviruses (family Geminiviridae) cause important diseases in many crops. In addition, many wild plants are reservoirs of begomoviruses, which are a potential menace for nearby crops. A novel begomovirus was isolated from a weed of the species Synedrella nodiflora (Compositae) exhibiting yellow vein symptoms in South India. This virus had a typical monopartite Old World begomovirus genome and was accompanied by a betasatellite. Sequence comparison revealed that this virus represents a new species in the genus Begomovirus. Recombination analysis showed that the novel begomovirus originated through recombination between the begomoviruses ageratum yellow vein Sri Lanka virus and tomato leaf curl Sri Lanka virus.

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants.

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.

Factors contributing to deletion within Mungbean yellow mosaic virus partial dimers in binary vectors used for agroinoculation.

Mungbean yellow mosaic virus-Vigna (MYMV) sequences cloned as partial dimers within the T-DNA of a binary vector were deleted at a high frequency upon conjugal mobilization from Escherichia coli into Agrobacterium tumefaciens. This deletion involving the genome-length viral DNA did not occur when the binary plasmid was inside E. coli and when the binary plasmid was introduced into Agrobacterium by electroporation. Deletions occurred in both DNA A and DNA B partial dimers. A minimum of 500-nt continuity on either side of the nonanucleotide in the duplicated common region is required for deletion. A. tumefaciens cells in which deletion was complete, grew as larger colonies reflecting a growth advantage. The small, slow-growing colonies eventually lost the genome-length viral sequences after a few more cycles of growth. Partial dimers in binary plasmids pGA472 and pBin19 with RK2 replicon underwent deletion while those in pPZP with pVS1 replicon did not undergo deletion. Deletion was observed in A. tumefaciens strains C58, A136, A348 and A281 with C58 chromosome background, but not in Ach5 and T37. Interestingly, deletion did not occur in A. tumefaciens strain AGL1 with a recA mutation in C58 chromosome, implying a clear role for recombination in deletion. These observations suggest the choice of Agrobacterium strains and binary vectors for agroinoculation of geminiviruses.

Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus.

Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.

Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes.

Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.

Analysis of an isolate of Mungbean yellow mosaic virus (MYMV) with a highly variable DNA B component.

One DNA A (KA30) and five different DNA B components (KA21, KA22, KA27, KA28 and KA34) of a geminivirus, Mungbean yellow mosaic virus-Vigna (MYMV-Vig) were cloned from a pooled sample of field-infected Vigna mungo plants from Vamban, South India. MYMV-Vig DNA A (KA30) and one of the DNA B components (KA27) exhibited 97% and 95% sequence identities, respectively, to those of MYMV reported from Thailand. However, the DNA B components KA21, KA22, KA28 and KA34 exhibited only 71 to 72% sequence identity to MYMV DNA B. Co-existence of multiple DNA B components in field-infected V. mungo was proved by Southern and PCR analyses. Each of the five DNA B components was infective together with the DNA A upon agroinoculation. Agroinoculation with mixed cultures of Agrobacterium with partial dimers of DNA A and all five DNA Bs proved that all five DNA B components can co-infect a single V. mungo plant.