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Gene silencing mediated by small noncoding RNAs (sRNAs) is a fundamental gene regulation mechanism in eukaryotes that broadly governs cellular processes. It has been established that sRNAs are critical regulators of plant growth, development, and antiviral defence, while accumulating studies support positive roles of sRNAs in plant defence against bacteria and eukaryotic pathogens such as fungi and oomycetes. Emerging evidence suggests that plant sRNAs move between species and function as antimicrobial agents against nonviral parasites. Multiple plant pathosystems have been shown to involve a similar exchange of small RNAs between species. Recent analysis about extracellular sRNAs shed light on the understanding of the selection and transportation of sRNAs moving from plant to parasites. In this review, we summarize current advances regarding the function and regulatory mechanism of plant endogenous small interfering RNAs (siRNAs) in mediating plant defence against pathogen intruders including viruses, bacteria, fungi, oomycetes, and parasitic plants. Beyond that, we propose potential mechanisms behind the sorting of sRNAs moving between species and the idea that engineering siRNA-producing loci could be a useful strategy to improve disease resistance of crops.

According to the less-is-more hypothesis, gene loss is an engine for evolutionary change. Loss-of-function (LoF) mutations resulting in the natural knockout of protein-coding genes not only provide information about gene function but also play important roles in adaptation and phenotypic diversification. Although the less-is-more hypothesis was proposed two decades ago, it remains to be explored on a large scale. In this study, we identified 60,819 LoF variants in 1071 Arabidopsis (Arabidopsis thaliana) genomes and found that 34% of Arabidopsis protein-coding genes annotated in the Columbia-0 genome do not have any LoF variants. We found that nucleotide diversity, transposable element density, and gene family size are strongly correlated with the presence of LoF variants. Intriguingly, 0.9% of LoF variants with minor allele frequency larger than 0.5% are associated with climate change. In addition, in the Yangtze River basin population, 1% of genes with LoF mutations were under positive selection, providing important insights into the contribution of LoF mutations to adaptation. In particular, our results demonstrate that LoF mutations shape diverse phenotypic traits. Overall, our results highlight the importance of the LoF variants for the adaptation and phenotypic diversification of plants.

// Wen Chen 1 , Wenrong He 3 , Hongbing Cai 4 , Bicheng Hu 1 , Caishang Zheng 2 , Xianliang Ke 2 , Li Xie 2 , Zhenhua Zheng 2 , Xinxing Wu 1 and Hanzhong Wang 2 1 State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China 2 State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China 3 Department of Gynaecology and Obstetrics, The First People’s Hospital of Jingzhou, Yangtze University, Jingzhou 434000, China 4 Department of Gynecologic Oncology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China Correspondence to: Xinxing Wu, email: wuxinxing9755@163.com Zhenhua Zheng, email: zhengzh@wh.iov.cn Keywords: A-to-I RNA editing, BLCAP gene, cervical carcinogenesis, STAT3, YXXQ motif Received: December 28, 2016 Accepted: March 21, 2017 Published: April 11, 2017 ABSTRACT Bladder cancer-associated protein (BLCAP) gene is a highly conserved gene with tumor-suppressor function in different carcinomas. It is also a novel ADAR-mediated editing substrate undergoes multiple A-to-I RNA editing events. Although the anti-tumorigenic role of BLCAP has been examined in preliminarily studies, the relationship between BLCAP function and A-to-I RNA editing in cervical carcinogenesis still require further exploration. Herein, we analyzed the coding sequence of BLCAP transcripts in 35 paired cervical cancer samples using high-throughput sequencing. Of note, editing levels of three novel editing sites were statistically different between cancerous and adjacent cervical tissues, and editing of these three sites was closely correlated. Moreover, two editing sites of BLCAP coding region were mapped-in the key YXXQ motif which can bind to SH2 domain of STAT3. Further studies revealed that BLCAP interacted with signal transducer and activator of transcription 3 (STAT3) and inhibited its phosphorylation, while A-to-I RNA editing of BLCAP lost the inhibition to STAT3 activation in cervical cancer cell lines. Our findings reveal that A-to-I RNA editing events alter the genetically coded amino acid in BLCAP YXXQ motif, which drive the progression of cervical carcinogenesis through regulating STAT3 signaling pathway.

Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5′−3′ RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5′−3′ exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation., Arabidopsis AGO1 binds both microRNA (miRNA) and endogenous small interfering RNA (siRNA). Here the authors show that 5′−3′ RNA degradation suppresses siRNA biogenesis from ribosomal RNA to prevent overloading of AGO1 with ribosomal siRNA and maintain miRNA-directed post translational gene silencing.

Plant development is regulated by both synergistic and antagonistic interactions of different phytohormones, including a complex crosstalk between ethylene and auxin. For instance, auxin and ethylene synergistically control primary root elongation and root hair formation. However, a lack of chemical agents that specifically modulate ethylene or auxin production has precluded precise delineation of the contribution of each hormone to root development. Here, we performed a chemical genetic screen based on the recovery of root growth in ethylene-related Arabidopsis mutants with constitutive "short root" phenotypes (eto1-2 and ctr1-1). We found that ponalrestat exposure recovers root elongation in these mutants in an ethylene signal-independent manner. Genetic and pharmacological investigations revealed that ponalrestat inhibits the enzymatic activity of the flavin-containing monooxygenase YUCCA, which catalyzes the rate-limiting step of the indole-3-pyruvic acid branch of the auxin biosynthesis pathway. In summary, our findings have identified a YUCCA inhibitor that may be useful as a chemical tool to dissect the distinct steps in auxin biosynthesis and in the regulation of root development.

Plants evolved an array of disease resistance genes (R genes) to fight pathogens. In the absence of pathogen infection, NBS-LRR genes, which comprise a major subfamily of R genes, are suppressed by a small RNA cascade involving microRNAs (miRNAs) that trigger the biogenesis of phased siRNAs (phasiRNAs) from R gene transcripts. However, whether or how R genes influence small RNA biogenesis is unknown. In this study, we isolate a mutant with global defects in the biogenesis of miRNAs and phasiRNAs in Arabidopsis thaliana and trace the defects to the over accumulation and nuclear localization of an R protein SNC1. We show that nuclear SNC1 represses the transcription of miRNA and phasiRNA loci, probably through the transcriptional corepressor TPR1. Intriguingly, nuclear SNC1 reduces the accumulation of phasiRNAs from three source R genes and concomitantly, the expression of a majority of the ~170R genes is up-regulated. Taken together, this study suggests an R gene-miRNA-phasiRNA regulatory module that amplifies plant immune responses., A small RNA-based signaling cascade prevents the induction of plant resistance genes (R-genes) in the absence of pathogen challenge. Here Cai et al. show that nuclear accumulation of the R protein SNC1 can activate immunity by suppressing small RNA production and releasing R-gene repression.

Plants evolved an array of disease resistance genes (R genes) to fight pathogens. In the absence of pathogen infection, NBS-LRR genes, which comprise a major subfamily of R genes, are suppressed by a small RNA cascade involving microRNAs (miRNAs) that trigger the biogenesis of phased siRNAs (phasiRNAs) from R gene transcripts. However, whether or how R genes influence small RNA biogenesis is unknown. In this study, we isolate a mutant with global defects in the biogenesis of miRNAs and phasiRNAs in Arabidopsis thaliana and trace the defects to the over accumulation and nuclear localization of an R protein SNC1. We show that nuclear SNC1 represses the transcription of miRNA and phasiRNA loci, probably through the transcriptional corepressor TPR1. Intriguingly, nuclear SNC1 reduces the accumulation of phasiRNAs from three source R genes and concomitantly, the expression of a majority of the ~170R genes is up-regulated. Taken together, this study suggests an R gene-miRNA-phasiRNA regulatory module that amplifies plant immune responses.

In plants, the cytokinin metabolic processes, including cytokinin biosynthesis, interconversion, inactivation, and degradation, play critical roles in the regulation of cytokinin homeostasis and plant development. Purine meta- bolic enzymes have been implied to catalyze the cytokinin interconversion in previous works. In this study, we report that Adenine Phosphoribosyl Transferase 1 (APT1) is the causal gene of the high-dose cytokinin-resistant mutants. APT1 catalyzes the cytokinin conversion from free bases to nucleotides, and is functionally predominant among the five members of the Arabidopsis Adenine Phosphoribosyl Transferase family. Loss of APT1 activity in plants leads to excess accumulation of cytokinin bases, thus evoking myriad cytokinin-regulated responses, such as delayed leaf senescence, anthocyanin accumulation, and downstream gene expression. Thus, our study defines APT1 as a key metabolic enzyme participating in the cytokinin inactivation by phosphoribosylation.

Ethylene is an important phytohormone that promotes the ripening of fruits and senescence of flowers thereby reducing their shelf lives. Specific ethylene biosynthesis inhibitors would help to decrease postharvest loss. Here, we identify pyrazinamide (PZA), a clinical drug used to treat tuberculosis, as an inhibitor of ethylene biosynthesis in Arabidopsis thaliana, using a chemical genetics approach. PZA is converted to pyrazinecarboxylic acid (POA) in plant cells, suppressing the activity of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), the enzyme catalysing the final step of ethylene formation. The crystal structures of Arabidopsis ACO2 in complex with POA or 2-Picolinic Acid (2-PA), a POA-related compound, reveal that POA/2-PA bind at the active site of ACO, preventing the enzyme from interacting with its natural substrates. Our work suggests that PZA and its derivatives may be promising regulators of plant metabolism, in particular ethylene biosynthesis., Ethylene is a plant hormone that promotes fruit ripening. Here, the authors identify pyrazinamide, a drug used to treat tuberculosis, as an inhibitor of ethylene biosynthesis in Arabidopsis thaliana and present the crystal structure of its active form (pyrazinecarboxylic acid) bound to ACC oxidase.

Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.

Phytohormone studies enlightened our knowledge of plant responses to various changes. To provide a systematic and comprehensive view of genes participating in plant hormonal regulation, an online accessible database Arabidopsis Hormone Database (AHD) has been developed, which is a collection of hormone related genes of the model organism Arabidopsis thaliana (AHRGs). Recently we updated our database from AHD to a new version AHD2.0 by adding several pronounced features: (i) updating our collection of AHRGs based on most recent publications as well as constructing elaborate schematic diagrams of each hormone biosynthesis and signaling pathways; (ii) adding orthologs of sequenced plants listed in OrthoMCL-DB to each AHRG in the updated database; (iii) providing predicted miRNA splicing site(s) for each AHRG; (iv) integrating genes that genetically interact with each AHRG according to literatures mining; (v) providing links to a powerful online analysis platform WebLab for the convenience of in-time bioinformatics analysis and (vi) providing links to widely used protein databases and integrating more expression profiling information that would facilitate users for a more systematic and integrative analysis related to phytohormone research.

Dark-grown Arabidopsis seedlings develop an apical hook when germinating in soil, which protects the cotyledons and apical meristematic tissues when protruding through the soil. Several hormones are reported to distinctly modulate this process. Previous studies have shown that ethylene and gibberellins (GAs) coordinately regulate the hook development, although the underlying molecular mechanism is largely unknown. Here we showed that GA(3) enhanced while paclobutrazol repressed ethylene- and EIN3-overexpression (EIN3ox)-induced hook curvature, and della mutant exhibited exaggerated hook curvature, which required an intact ethylene signaling pathway. Genetic study revealed that GA-enhanced hook development was dependent on HOOKLESS 1 (HLS1), a central regulator mediating the input of the multiple signaling pathways during apical hook development. We further found that GA(3) induced (and DELLA proteins repressed) HLS1 expression in an ETHYLENE INSENSITIVE 3/EIN3-LIKE 1 (EIN3/EIL1)-dependent manner, whereby EIN3/EIL1 activated HLS1 transcription by directly binding to its promoter. Additionally, DELLA proteins were found to interact with the DNA-binding domains of EIN3/EIL1 and repress EIN3/EIL1-regulated HLS1 expression. Treatment with naphthylphthalamic acid, a polar auxin transport inhibitor, repressed the constitutively exaggerated hook curvature of EIN3ox line and della mutant, supporting that auxin functions downstream of the ethylene and GA pathways in hook development. Taken together, our results identify EIN3/EIL1 as a new class of DELLA-associated transcription factors and demonstrate that GA promotes apical hook formation in cooperation with ethylene partly by inducing the expression of HLS1 via derepression of EIN3/EIL1 functions.

The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

Plant responses to ethylene are mediated by regulation of EBF1/2-dependent degradation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Here, we report that the level of EIL1 protein is upregulated by ethylene through an EBF1/2-dependent pathway. Genetic analysis revealed that EIL1 and EIN3 cooperatively but differentially regulate a wide array of ethylene responses, with EIL1 mainly inhibiting leaf expansion and stem elongation in adult plants and EIN3 largely regulating a multitude of ethylene responses in seedlings. When EBF1 and EBF2 are disrupted, EIL1 and EIN3 constitutively accumulate in the nucleus and remain unresponsive to exogenous ethylene application. Further study revealed that the levels of EBF1 and EBF2 proteins are downregulated by ethylene and upregulated by silver ion and MG132, suggesting that ethylene stabilizes EIN3/EIL1 by promoting EBF1 and EBF2 proteasomal degradation. Also, we found that EIN2 is indispensable for mediating ethylene-induced EIN3/EIL1 accumulation and EBF1/2 degradation, whereas MKK9 is not required for ethylene signal transduction, contrary to a previous report. Together, our studies demonstrate that ethylene similarly regulates EIN3 and EIL1, the two master transcription factors coordinating myriad ethylene responses, and clarify that EIN2 but not MKK9 is required for ethylene-induced EIN3/EIL1 stabilization. Our results also reveal that EBF1 and EBF2 act as essential ethylene signal transducers that by themselves are subject to proteasomal degradation.