OsmiR319-OsPCF5 modulate resistance to brown planthopper in rice through association with MYB proteins.

BACKGROUND: The brown planthopper (BPH) is a kind of piercing-sucking insect specific to rice, with the damage tops the list of pathogens and insects in recent years. microRNAs (miRNAs) are pivotal regulators of plant-environment interactions, while the mechanism underlying their function against insects is largely unknown. RESULTS: Here, we confirmed that OsmiR319, an ancient and conserved miRNA, negatively regulated resistance to BPHs, with overexpression of OsmiR319 susceptible to BPH, while suppression of OsmiR319 resistant to BPH in comparison with wild type. Meanwhile, we identified several targets of OsmiR319 that may mediate BPH resistance. Among them, OsPCF5 was the most obviously induced by BPH feeding, and over expression of OsPCF5 was resistance to BPH. In addition, various biochemical assays verified that OsPCF5 interacted with several MYB proteins, such as OsMYB22, OsMYB30, and OsMYB30C.Genetically, we revealed that both OsMYB22 and OsMYB30C positively regulated BPH resistance. Genetic interaction analyses confirmed that OsMYB22 and OsMYB30C both function in the same genetic pathway with OsmiR319b to mediate BPH resistance. CONCLUSIONS: Altogether, we revealed that OsPCF5 regulates BPH resistance via association with several MYB proteins downstream of OsmiR319, these MYB proteins might function as regulators of BPH resistance through regulating the phenylpropane synthesis.

A novel transcriptional repressor complex MYB22-TOPLESS-HDAC1 promotes rice resistance to brown planthopper by repressing F3'H expression.

The brown planthopper (BPH) is the most destructive pest of rice. The MYB transcription factors are vital for rice immunity, but most are activators. Although MYB22 positively regulates rice resistance to BPH and has an EAR motif associated with active repression, it remains unclear whether it is a transcriptional repressor affecting rice-BPH interaction. Genetic analyses revealed that MYB22 regulates rice resistance to BPH via its EAR motif. Several biochemical experiments (e.g. transient transcription assay, Y2H, LCA, and BiFC) indicated that MYB22 is a transcriptional repressor that interacts with the corepressor TOPLESS via its EAR motif and recruits HDAC1 to form a tripartite complex. Flavonoid-3'-hydroxylase (F3'H) is a flavonoid biosynthesis pathway-related gene that negatively regulates rice resistance to BPH. Based on a bioinformatics analysis and the results of EMSA and transient transcription assays, MYB22 can bind directly to the F3'H promoter and repress gene expression along with TOPLESS and HDAC1. We revealed a transcriptional regulatory mechanism influencing the rice-BPH interaction that differs from previously reported mechanisms. Specifically, MYB22-TOPLESS-HDAC1 is a novel transcriptional repressor complex with components that synergistically and positively regulate rice resistance to BPH through the transcriptional repression of F3'H.

OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408.

microRNAs (miRNAs) are promising targets for crop improvement of complex agricultural traits. Coordinated activity between/among different miRNAs may fine-tune specific developmental processes in diverse organisms. Grain size is a main factor determining rice (Oryza sativa L.) crop yield, but the network of miRNAs influencing this trait remains uncharacterized. Here we show that sequestering OsmiR396 through target mimicry (MIM396) can substantially increase grain size in several japonica and indica rice subspecies and in plants with excessive tillers and a high panicle density. Thus, OsmiR396 has a major role related to the regulation of rice grain size. The grain shape of Growth Regulating Factor8 (OsGRF8)-overexpressing transgenic plants was most similar to that of MIM396 plants, suggesting OsGRF8 is a major mediator of OsmiR396 in grain size regulation. A miRNA microarray analysis revealed changes to the expression of many miRNAs, including OsmiR408, in the MIM396 plants. Analyses of gene expression patterns and functions indicated OsmiR408 is an embryo-specific miRNA that positively regulates grain size. Silencing OsmiR408 expression (miR408KO) using CRISPR technology resulted in small grains. Moreover, we revealed the direct regulatory effects of OsGRF8 on OsMIR408 expression. A genetic analysis further showed that the large-grain phenotype of MIM396 plants could be complemented by miR408KO. Also, several hormone signaling pathways might be involved in the OsmiR396/GRF-meditated grain size regulation. Our findings suggest that genetic regulatory networks comprising various miRNAs, such as OsmiR396 and OsmiR408, may be crucial for controlling rice grain size. Furthermore, the OsmiR396/GRF module may be important for breeding new high-yielding rice varieties.

Identification of the rice genes and metabolites involved in dual resistance against brown planthopper and rice blast fungus.

Brown planthopper (BPH) and blast disease jointly or individually cause big yield losses every year. To identify genes and metabolites with potential contributions to the dual resistance against both biotic-stress factors, we carried out a transcriptome and metabolome analysis for susceptible and resistant rice varieties after BPH and rice blast infestations. Coexpression network analysis identified a modular pattern that had the highest correlation coefficients (0.81) after the BPH and rice blast (-0.81) treatments. In total, 134 phenylpropanoid biosynthesis pathway-related genes were detected in this group. We found that the flavanone 3-hydroxylase gene (OsF3H) had opposite expression trends in response to BPH and rice blast infestations whereas the OsF3'H had similar expression patterns. Genetics analysis confirmed that the OsF3H gene knockdown lines demonstrated the opposite resistance phenotypes against BPH and rice blast, whereas the OsF3'H knockout lines enhanced rice resistance against both pests. Consistently, our metabolomics analysis identified the metabolite eriodictyol, one putative essential product of these two genes, that was more highly accumulated in the resistant rice variety of RHT than in the susceptible variety MDJ. This study highlights a useful strategy for identifying more genes and metabolites that have potential synergistic effects on rice against to multiple biotic stresses.

Identification of GROWTH-REGULATING FACTOR transcription factors in lettuce (Lactuca sativa) genome and functional analysis of LsaGRF5 in leaf size regulation.

BACKGROUND: GROWTH-REGULATING FACTORs (GRFs), a type of plant-specific transcription factors, play important roles in regulating plant growth and development. Although GRF gene family has been identified in various plant species, a genome-wide analysis of this family in lettuce (Lactuca sativa L.) has not been reported yet. RESULTS: Here we identified 15 GRF genes in lettuce and performed comprehensive analysis of them, including chromosomal locations, gene structures, and conserved motifs. Through phylogenic analysis, we divided LsaGRFs into six groups. Transactivation assays and subcellular localization of LsaGRF5 showed that this protein is likely to act as a transcriptional factor in the cell nucleus. Furthermore, transgenic lettuce lines overexpressing LsaGRF5 exhibited larger leaves, while smaller leaves were observed in LsaMIR396a overexpression lines, in which LsaGRF5 was down-regulated. CONCLUSIONS: These results in lettuce provide insight into the molecular mechanism of GRF gene family in regulating leaf growth and development and foundational information for genetic improvement of the lettuce variations specialized in leaf character.

Novel crosstalk between ethylene- and jasmonic acid-pathway responses to a piercing-sucking insect in rice.

Ethylene (ET) and jasmonic acid (JA) play important roles in plant defenses against biotic stresses. Crosstalk between JA and ET has been well studied in mediating pathogen resistance, but its roles in piercing-sucking insect resistance are unclear. The brown planthopper (BPH; Nilaparvata lugens) is the most notorious piercing-sucking insect specific to rice (Oryza sativa) that severely affects yield. A genetic analysis revealed that OsEBF1 and OsEIL1, which are in the ET signaling pathway, positively and negatively regulated BPH resistance, respectively. Molecular and biochemical analyses revealed direct interactions between OsEBF1 and OsEIL1. OsEBF1, an E3 ligase, mediated the degradation of OsEIL1 through the ubiquitination pathway, indicating the negative regulation of the ET-signaling pathway in response to BPH infestation. An RNA sequencing analysis revealed that a JA biosynthetic pathway-related gene, OsLOX9, was downregulated significantly in the oseil1 mutant. Biochemical analyses, including yeast one-hybrid, dual luciferase, and electrophoretic mobility shift assay, confirmed the direct regulation of OsLOX9 by OsEIL1. This study revealed the synergistic and negative regulation of JA and ET pathways in response to piercing-sucking insect attack. The synergistic mechanism was realized by transcriptional regulation of OsEIL1 on OsLOX9. OsEIL1-OsLOX9 is a novel crosstalk site in these two phytohormone signaling pathways.

The OsmiR396-OsGRF8-OsF3H-flavonoid pathway mediates resistance to the brown planthopper in rice (Oryza sativa).

Multi-functional microRNAs (miRNAs) are emerging as key modulators of plant-pathogen interactions. Although the involvement of some miRNAs in plant-insect interactions has been revealed, the underlying mechanisms are still elusive. The brown planthopper (BPH) is the most notorious rice (Oryza sativa)-specific insect that causes severe yield losses each year and requires urgent biological control. To reveal the miRNAs involved in rice-BPH interactions, we performed miRNA sequencing and identified BPH-responsive OsmiR396. Sequestering OsmiR396 by overexpressing target mimicry (MIM396) in three genetic backgrounds indicated that OsmiR396 negatively regulated BPH resistance. Overexpression of one BPH-responsive target gene of OsmiR396, growth regulating factor 8 (OsGRF8), showed resistance to BPH. Furthermore, the flavonoid contents increased in both the OsmiR396-sequestered and the OsGRF8 overexpressing plants. By analysing 39 natural rice varieties, the elevated flavonoid contents were found to correlate with enhanced BPH resistance. Artificial applications of flavonoids to wild type (WT) plants also increased resistance to BPH. A BPH-responsive flavanone 3-hydroxylase (OsF3H) gene in the flavonoid biosynthetic pathway was proved to be directly regulated by OsGRF8. A genetic functional analysis of OsF3H revealed its positive role in mediating both the flavonoid contents and BPH resistance. And analysis of the genetic correlation between OsmiR396 and OsF3H showed that down-regulation of OsF3H complemented the BPH resistance characteristic and simultaneously decreased the flavonoid contents of the MIM396 plants. Thus, we revealed a new BPH resistance mechanism mediated by the OsmiR396-OsGRF8-OsF3H-flavonoid pathway. Our study suggests potential applications of miRNAs in BPH resistance breeding.

An AT-hook protein DEPRESSED PALEA1 physically interacts with the TCP Family transcription factor RETARDED PALEA1 in rice.

The cereal crops (such as rice and maize) which belong to the grass family, are the most important grain crops for human beings, and the development of their flower and inflorescence architecture has attracted extensive attention. Although multiple genes involved in the regulation of floral and inflorescence organogenesis have been identified, the underlying molecular mechanisms are largely unknown. Previously, we identified rice depressed palea1 (dp1) mutants with defects in main structure of palea and its enhancer RETARDED PALEA1 (REP1). DP1 is an AT-hook protein while REP1 is a TCP transcription factor, both of which are important regulators of palea development. However, the relationship of these two proteins has not been elucidated yet. Here, we demonstrated that DP1 interacts physically with REP1 both in yeast and in rice protoplasts. Considering the close phylogenetic relationship between maize and rice, we further hypothesize that their orthologs in maize, BARREN STALK FASTIGIATE (BAF1) and BRANCH ANGLE DEFECTIVE 1 (BAD1), may interact physically. Subsequently, we verified their physical interaction, indicating that the interaction between AT-hook proteins and TCP proteins is conserved in rice and maize. Our findings may reveal a novel molecular mechanism of floral and inflorescence development in grasses.

Calcium Pumps and Interacting BON1 Protein Modulate Calcium Signature, Stomatal Closure, and Plant Immunity.

Calcium signaling is essential for environmental responses including immune responses. Here, we provide evidence that the evolutionarily conserved protein BONZAI1 (BON1) functions together with autoinhibited calcium ATPase10 (ACA10) and ACA8 to regulate calcium signals in Arabidopsis. BON1 is a plasma membrane localized protein that negatively regulates the expression of immune receptor genes and positively regulates stomatal closure. We found that BON1 interacts with the autoinhibitory domains of ACA10 and ACA8, and the aca10 loss-of-function (LOF) mutants have an autoimmune phenotype similar to that of the bon1 LOF mutants. Genetic evidences indicate that BON1 positively regulates the activities of ACA10 and ACA8. Consistent with this idea, the steady level of calcium concentration is increased in both aca10 and bon1 mutants. Most strikingly, cytosolic calcium oscillation imposed by external calcium treatment was altered in aca10, aca8, and bon1 mutants in guard cells. In addition, calcium- and pathogen-induced stomatal closure was compromised in the aca10 and bon1 mutants. Taken together, this study indicates that ACA10/8 and BON1 physically interact on plasma membrane and function in the generation of cytosol calcium signatures that are critical for stomatal movement and impact plant immunity.

Modulation of plant architecture by the miR156f-OsSPL7-OsGH3.8 pathway in rice.

Tiller number and plant height are two of the main features of plant architecture that directly influence rice yield. Auxin and miR156, an extensively studied small RNA (smRNA), are both broadly involved in plant development and physiology, suggesting a possible relationship between the two. In this study, we identified a rice T-DNA insertion cluster and dwarf (cd) mutant that has an increased tiller number and reduced plant height. The T-DNA insertion was in close proximity to the miR156f gene and was associated with its up-regulation. Plants overexpressing miR156f resembled the cd mutant. In contrast, plants overexpressing an miR156f target mimic (MIM156fOE) had a reduced tiller number and increased height. Genetic analysis showed that OsSPL7 is a target of miR156f that regulates plant architecture. Plants overexpressing OsSPL7 had a reduced tiller number, while OsSPL7 RNAi plants had an increased tiller number and a reduced height. We also found that OsSPL7 binds directly to the OsGH3.8 promoter to regulate its transcription. Overexpression of OsGH3.8 and OsGH3.8 RNAi partially complemented the MIM156fOE and cd mutant phenotypes, respectively. Our combined data show that the miR156f-OsSPL7-OsGH3.8 pathway regulates tiller number and plant height in rice, and this pathway may allow crosstalk between miR156 and auxin.

OsEXPA10 mediates the balance between growth and resistance to biotic stress in rice.

KEY MESSAGE: OsEXPA10 gene coordinates the balance between rice development and biotic resistance. Expansins are proteins that can loosen the cell wall. Previous studies have indicated that expansin-encoding genes were involved in defense against abiotic stress, but little is known about the involvement of expansins in biotic stress. Brown planthopper (BPH) is one of the worst insect pests of rice in the Asia-Pacific planting area, and many efforts have been made to identify and clone BPH-resistance genes for use in breeding resistant cultivars. At the same time, rice blast caused by Magnaporthe grisea is one of the three major diseases that severely affect rice production worldwide. Here, we demonstrated that one rice expansin-encoding gene, OsEXPA10, functions in both rice growth and biotic resistance. Over expression of OsEXPA10 improved rice growth but also increased susceptibility to BPH infestation and blast attack, while knock-down OsEXPA10 gene expression resulted in reduced plant height and grain size, but also increased resistance to BPH and the blast pathogen. These results imply that OsEXPA10 mediates the balance between rice development and biotic resistance.

The Arabidopsis Chromatin-Remodeling Factor CHR5 Regulates Plant Immune Responses and Nucleosome Occupancy.

ATP-dependent chromatin-remodeling factors use the energy of ATP hydrolysis to alter the structure of chromatin and are important regulators of eukaryotic gene expression. One such factor encoded by CHR5 (Chromatin-Remodeling Factor 5) in Arabidopsis (Arabidopsis thaliana) was previously found to be involved in regulation of growth and development. Here we show that CHR5 is required for the up-regulation of the intracellular immune receptor gene SNC1 (SUPPRESSOR OF npr1-1, CONSTITUTIVE1) and consequently the autoimmunity induced by SNC1 up-regulation. CHR5 functions antagonistically with another chromatin-remodeling gene DDM1 (DECREASED DNA METHYLATION 1) and independently with a histone mono-ubiquitinase HUB1 (HISTONE MONOUBIQUITINATION 1) in SNC1 regulation. In addition, CHR5 is a positive regulator of SNC1-independent plant immunity against the bacterial pathogen Pseudomonas syringae. Furthermore, the chr5 mutant has increased nucleosome occupancy in the promoter region relative to the gene body region at the whole-genome level, suggesting a global role for CHR5 in remodeling nucleosome occupancy. Our study thus establishes CHR5 as a positive regulator of plant immune responses including the expression of SNC1 and reveals a role for CHR5 in nucleosome occupancy which probably impacts gene expression genome wide.

Albino Leaf1 That Encodes the Sole Octotricopeptide Repeat Protein Is Responsible for Chloroplast Development.

Chloroplast, the photosynthetic organelle in plants, plays a crucial role in plant development and growth through manipulating the capacity of photosynthesis. However, the regulatory mechanism of chloroplast development still remains elusive. Here, we characterized a mutant with defective chloroplasts in rice (Oryza sativa), termed albino leaf1 (al1), which exhibits a distinct albino phenotype in leaves, eventually leading to al1 seedling lethality. Electronic microscopy observation demonstrated that the number of thylakoids was reduced and the structure of thylakoids was disrupted in the al1 mutant during rice development, which eventually led to the breakdown of chloroplast. Molecular cloning revealed that AL1 encodes the sole octotricopeptide repeat protein (RAP) in rice. Genetic complementation of Arabidopsis (Arabidopsis thaliana) rap mutants indicated that the AL1 protein is a functional RAP. Further analysis illustrated that three transcript variants were present in the AL1 gene, and the altered splices occurred at the 3' untranslated region of the AL1 transcript. In addition, our results also indicate that disruption of the AL1 gene results in an altered expression of chloroplast-associated genes. Consistently, proteomic analysis demonstrated that the abundance of photosynthesis-associated proteins is altered significantly, as is that of a group of metabolism-associated proteins. More specifically, we found that the loss of AL1 resulted in altered abundances of ribosomal proteins, suggesting that RAP likely also regulates the homeostasis of ribosomal proteins in rice in addition to the ribosomal RNA. Taken together, we propose that AL1, particularly the AL1a and AL1c isoforms, plays an essential role in chloroplast development in rice.

Opposing Effects on Two Phases of Defense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis.

The plant immune system consists of multiple layers of responses targeting various phases of pathogen infection. Here, we provide evidence showing that two responses, one controlling stomatal closure and the other mediated by intracellular receptor proteins, can be regulated by the same proteins but in an antagonistic manner. The HEAT SHOCK COGNATE70 (HSC70), while previously known as a negative regulator of stomatal closure, is a positive regulator of immune responses mediated by the immune receptor protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE1 (SNC1) as well as basal defense responses. In contrast to HSC70, a calcium-binding protein, BONZAI1 (BON1), promotes abscisic acid- and pathogen-triggered stomatal closure in addition to and independent of its previously known negative role in SNC1 regulation. BON1 likely regulates stomatal closure through activating SUPPESSOR OF THE G2 ALLELE OF SKP1 VARIANT B and inhibiting HSC70. New functions of BON1 and HSC70 identified in this study thus reveal opposite effects of each of them on immunity. The opposing roles of these regulators at different phases of plant immune responses exemplify the complexity in immunity regulation and suggest that immune receptors may guard positive regulators functioning at stomatal closure control.

Monoubiquitination of histone 2B at the disease resistance gene locus regulates its expression and impacts immune responses in Arabidopsis.

Disease resistance (R) genes are key components in plant immunity. Here, we show that Arabidopsis (Arabidopsis thaliana) E3 ubiquitin ligase genes HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 regulate the expression of R genes SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1) and RESISTANCE TO PERONOSPORA PARASITICA4. An increase of SNC1 expression induces constitutive immune responses in the bonzai1 (bon1) mutant, and the loss of HUB1 or HUB2 function reduces SNC1 up-regulation and suppresses the bon1 autoimmune phenotypes. HUB1 and HUB2 mediate histone 2B (H2B) monoubiquitination directly at the SNC1 R gene locus to regulate its expression. In addition, SNC1 and HUB1 transcripts are moderately up-regulated by pathogen infection, and H2B monoubiquitination at SNC1 is enhanced by pathogen infection. Together, this study indicates that H2B monoubiquitination at the R gene locus regulates its expression and that this histone modification at the R gene locus has an impact on immune responses in plants.

The F-box protein CPR1/CPR30 negatively regulates R protein SNC1 accumulation.

Disease resistance (R) proteins, as central regulators of plant immunity, are tightly regulated for effective defense responses and to prevent constitutive defense activation under non-pathogenic conditions. Here we report the identification of an F-box protein CPR1/CPR30 as a negative regulator of an R protein SNC1 likely through SCF (Skp1-cullin-F-box) mediated protein degradation. The cpr1-2 (cpr30-1) loss-of-function mutant has constitutive defense responses, and it interacts synergistically with a gain-of function mutant snc1-1 and a bon1-1 mutant where SNC1 is upregulated. The loss of SNC1 function suppresses the mutant phenotypes of cpr1-2 and cpr1-2 bon1-1, while overexpression of CPR1 rescues mutant phenotypes of both bon1-1 and snc1-1. Furthermore, the amount of SNC1 protein is upregulated in the cpr1-2 mutant and down-regulated when CPR1 is overexpressed. The regulation of SNC1 by CPR1 is dependent on the 26S proteosome as a protease inhibitor MG132 stabilizes SNC1 and reverses the effect of CPR1 on SNC1. Interestingly, CPR1 is induced after infection of both virulent and avirulent pathogens similarly to the other negative defense regulator BON1. Thus, this study reveals a new mechanism in R protein regulation likely through protein degradation and suggests negative regulation as a critical component in fine control of plant immunity.

OsmiR159 Modulate BPH Resistance Through Regulating G-Protein γ Subunit GS3 Gene in Rice.

Brown planthopper (BPH) is the most destructive insect pest to rice that causes tremendous yield loss each year in rice planting Asia and South-East Asia areas. Compared with traditional chemical-based treatment, utilization of plant endogenous resistance is a more effective and environmental-friendly way for BPH control. Accordingly, quite a few quantitative trait loci (QTLs) for BPH resistance were cloned using forward genetics. However, BPH is apt to change quickly into new biotypes to overcome plant resistance, therefore, new resistance resources and genes are continuously needed. miRNAs are important regulators in both plant development and physiological regulation including immunity, and might be used as effective supplements for BPH resistance QTLs. miR159 is an ancient and conserved miRNA. In this study, we found that each OsMIR159 gene in rice responded to BPH feeding very obviously, and genetic function assay proved them to negatively regulate BPH resistance, with STTM159 showing resistance to BPH, and over expression of OsmiR159d susceptible to BPH. One target genes of OsmiR159, OsGAMYBL2, positively regulated BPH resistance. Further biochemical studies revealed that OsGAMYBL2 could directly bind to the promoter of G-protein γ subunit encoding GS3 gene and repress its expression. And genetically, GS3 responded to BPH feeding promptly and negatively regulated BPH resistance, GS3 over expression plants were susceptible to BPH, while GS3 knock-out plants were resistant to BPH. Thus, we identified new function of OsmiR159-OsGAMYBL2 in mediating BPH response, and revealed a new OsmiR159-G protein pathway that mediates BPH resistance in rice.

Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice.

Planthoppers are highly destructive pests in crop production worldwide. Brown planthopper (BPH) causes the most serious damage of the rice crop globally among all rice pests. Growing resistant varieties is the most effective and environment-friendly strategy for protecting the crop from BPH. More than 19 BPH-resistance genes have been reported and used to various extents in rice breeding and production. In this study, we cloned Bph14, a gene conferring resistance to BPH at seedling and maturity stages of the rice plant, using a map-base cloning approach. We show that Bph14 encodes a coiled-coil, nucleotide-binding, and leucine-rich repeat (CC-NB-LRR) protein. Sequence comparison indicates that Bph14 carries a unique LRR domain that might function in recognition of the BPH insect invasion and activating the defense response. Bph14 is predominantly expressed in vascular bundles, the site of BPH feeding. Expression of Bph14 activates the salicylic acid signaling pathway and induces callose deposition in phloem cells and trypsin inhibitor production after planthopper infestation, thus reducing the feeding, growth rate, and longevity of the BPH insects. Our work provides insights into the molecular mechanisms of rice defense against insects and facilitates the development of resistant varieties to control this devastating insect.

Evolutionary characterization of miR396s in Poaceae exemplified by their genetic effects in wheat and maize.

MiR396s play important roles in regulating plant growth and stress response, and great potential for crop yield promotion was anticipated. For more comprehensive and precise understanding of miR396s in Poaceae, we analyzed the phylogenetic linkage, gene expression, and chromosomal distribution of miR396s in this study. Although the mature miR396s' sequences were mostly conserved, differential expression patterns and chromosomal distribution were found among Poaceae species including the major cereal crops rice, wheat, and maize. Consistently, in comparison with rice, wheat and maize plants transformed with the target mimicry construct of miR396 (MIM396) exhibited differential effects on grain size and disease resistance. While the TaMIM396 plants showed increased grain size, panicle length and sensitivity to B. graminis, the ZmMIM396 plants didn't show obvious changes in grain size and disease resistance. In Addition, several GROWTH-REGULATING FACTOR (GRF) genes in wheat and maize were repressed by miR396s, which could be reversed by MIM396, confirming the conserved regulatory roles of miR396 on GRFs. While providing new solution to enhance grain yield in wheat and revealing potential regulatory variations of miR396s in controlling grain size and disease resistance in different crops, this study gives clues to further explore miR396s' functions in other Poaceae species.

Overexpression of Osta-siR2141 caused abnormal polarity establishment and retarded growth in rice.

Small RNAs (smRNAs) including miRNAs and siRNAs are critical for gene regulation and plant development. Among the highly diverse siRNAs, trans-acting siRNAs (ta-siRNAs) have been shown to be plant-specific. In Arabidopsis, eight TAS loci belonging to four families (TAS1, TAS2, TAS3, and TAS4) have been identified, and bioinformatics analysis reveals that the sequence of TAS3 is highly conserved in plants. In this study, the function of TAS3 ta-siRNA (tasiR-ARF) has been revealed in rice (Oryza sativa L.) on polarity establishment and stage transition from vegetative to reproductive development by over-expressing Osta-siR2141. Osta-siR2141 replaced miR390 in the miR390 backbone for ectopic expression in rice, and overexpression of Osta-siR2141 caused disturbed vascular bundle development and adaxialization in polarity establishment. Transgenic lines also displayed abnormal shoot apical meristems (SAMs) and retarded growth at the vegetative stage. Molecular analysis revealed that overexpression of Osta-siR2141 resulted in the down-regulation of miR166 and the up-regulation of class III homeodomain-leucine zipper genes (HD-ZIPIIIs) in the vegetative stage but not in the reproductive stage. Moreover, overexpression of Osta-siR2141 in Arabidopsis disturbed polarity establishment and retarded stage transition, suggesting that tasiR-ARF was functionally conserved in rice and Arabidopsis.