A Genetic Screen for Impaired Systemic RNAi Highlights the Crucial Role of DICER-LIKE 2.

Posttranscriptional gene silencing (PTGS) of transgenes involves abundant 21-nucleotide small interfering RNAs (siRNAs) and low-abundance 22-nucleotide siRNAs produced from double-stranded RNA (dsRNA) by DCL4 and DCL2, respectively. However, DCL2 facilitates the recruitment of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to ARGONAUTE 1-derived cleavage products, resulting in more efficient amplification of secondary and transitive dsRNA and siRNAs. Here, we describe a reporter system where RDR6-dependent PTGS is initiated by restricted expression of an inverted-repeat dsRNA specifically in the Arabidopsis (Arabidopsis thaliana) root tip, allowing a genetic screen to identify mutants impaired in RDR6-dependent systemic PTGS. Our screen identified dcl2 but not dcl4 mutants. Moreover, grafting experiments showed that DCL2, but not DCL4, is required in both the source rootstock and the recipient shoot tissue for efficient RDR6-dependent systemic PTGS. Furthermore, dcl4 rootstocks produced more DCL2-dependent 22-nucleotide siRNAs than the wild type and showed enhanced systemic movement of PTGS to grafted shoots. Thus, along with its role in recruiting RDR6 for further amplification of PTGS, DCL2 is crucial for RDR6-dependent systemic PTGS.

VpWRKY3, a biotic and abiotic stress-related transcription factor from the Chinese wild Vitis pseudoreticulata.

Chinese wild grapevine Vitis pseudoreticulata accession 'Baihe-35-1' is identified as the precious resource with multiple resistances to pathogens. A directional cDNA library was constructed from the young leaves inoculated with Erysiphe necator. A total of 3,500 clones were sequenced, yielding 1,727 unigenes. Among them, 762 unigenes were annotated and classified into three classes, respectively, using Gene Ontology, including 22 ESTs related to transcription regulator activity. A novel WRKY transcription factor was isolated from the library, and designated as VpWRKY3 (GenBank Accession No. JF500755). The full-length cDNA is 1,280 bp, encoding a WRKY protein of 320 amino acids. VpWRKY3 is localized to nucleus and functions as a transcriptional activator. QRT-PCR analysis showed that the VpWRKY3 specifically accumulated in response to pathogen, salicylic acid, ethylene and drought stress. Overexpression of VpWRKY3 in tobacco increased the resistance to Ralstonia solanacearum, indicating that VpWRKY3 participates in defense response. Furthermore, VpWRKY3 is also involved in abscisic acid signal pathway and salt stress. This experiment provided an important basis for understanding the defense mechanisms mediated by WRKY genes in China wild grapevine. Generation of the EST collection from the cDNA library provided valuable information for the grapevine breeding. Key message We constructed a cDNA library from Chinese wild grapevine leaves inoculated with powdery mildew. VpWRKY3 was isolated and demonstrated that it was involved in biotic and abiotic stress responses.

Isolation and functional characterization of a transcription factor VpNAC1 from Chinese wild Vitis pseudoreticulata.

NAC (for NAM, ATAF1, 2, and CUC2) family genes encode plant-specific transcription factors that play important roles in plant development regulation and in abiotic and biotic stresses. However, the function of NAC genes in grapevines is not clear. A novel NAC transcription factor, designated as VpNAC1, was isolated from Chinese wild Vitis pseudoreticulata. It belongs to the TERN subgroup and is a nuclear-targeting protein and functions as a transcriptional activator. Moreover, VpNAC1 was induced by the fungus Erysiphe necator and the exogenous hormones, particularly salicylic acid, methyl jasmonate and ethylene. Over-expression of VpNAC1 in tobacco plants enhanced their resistance to Erysiphe cichoracearum and Phytophthora parasitica var. nicotianae Tucker. These results suggest that VpNAC1 acts as a positive regulator in biotic stresses.

Contrasting epigenetic control of transgenes and endogenous genes promotes post-transcriptional transgene silencing in Arabidopsis.

Transgenes that are stably expressed in plant genomes over many generations could be assumed to behave epigenetically the same as endogenous genes. Here, we report that whereas the histone H3K9me2 demethylase IBM1, but not the histone H3K4me3 demethylase JMJ14, counteracts DNA methylation of Arabidopsis endogenous genes, JMJ14, but not IBM1, counteracts DNA methylation of expressed transgenes. Additionally, JMJ14-mediated specific attenuation of transgene DNA methylation enhances the production of aberrant RNAs that readily induce systemic post-transcriptional transgene silencing (PTGS). Thus, the JMJ14 chromatin modifying complex maintains expressed transgenes in a probationary state of susceptibility to PTGS, suggesting that the host plant genome does not immediately accept expressed transgenes as being epigenetically the same as endogenous genes.

Stronger Increases in Cognitive Functions among Socio-Economically Disadvantaged Older Adults in China: A Longitudinal Analysis with Multiple Birth Cohorts.

Highly variable changes in cognitive functions occur as people get older, and socio-economically disadvantaged older adults are more likely to suffer from cognitive decline. This study aims to identify the longitudinal trend in cognitive functions among different socio-economic groups of older adults. The Chinese Longitudinal Healthy Longevity Survey (CLHLS) followed up 32,323 individuals aged 65 years and older over a 12-year period. A series of mixed-effects models was used to explicitly assess cohort trend and its socio-economic disparity in the cognitive functions of older adults. Scores for significant increase in cognitive functions by birth cohort were smaller by 0.49, 0.28, and 0.64 among older adults with more educational experience, a lower household income, or economic dependence relative to their counterparts. Scores for differences in cognitive function between older adults with higher and lower incomes were smaller by 0.46 among those living in urban areas than among those living in rural areas. Although there were larger cohort growth trends in cognitive functions among older adults with lower educational attainment, lower household income, and who were economically dependent, effective public intervention targeting these socio-economically disadvantaged populations is still necessary.

Socioeconomic disparities in leisure activities over the life course of the oldest-old in China.

OBJECTIVE: To identify the socioeconomic factors leading to disparities in participation in leisure activities over the life course among Chinese oldest-old people. METHODS: Data are from the Chinese Longitudinal Healthy Longevity Survey (CLHLS), a sample of adults aged 80 years or older in the period 1998-2012, featuring 38 312 individuals (58 945 observations). RESULTS: The younger birth cohorts were found to have significantly higher levels of participation in leisure activities, with a non-linear decrease in age observed for oldest-old men and women. Higher participation levels were significantly related to higher education levels, economic independence and non-manual occupations before retirement among the Chinese oldest-old. CONCLUSIONS: Leisure activity participation has increased substantially in recent cohorts of Chinese oldest-olds. Those with lower socioeconomic status may represent a disadvantaged population in terms of participation in leisure activities over their life course.

Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes.

BACKGROUND: Forward genetic screens are a powerful approach for identifying the genes contributing to a trait of interest. However, mutants arising in genes already known can obscure the identification of new genes contributing to the trait. Here, we describe a strategy called Candidate gene-Sequencing (Can-Seq) for rapidly identifying and filtering out mutants carrying new alleles of known and candidate genes. RESULTS: We carried out a forward genetic screen and identified 40 independent Arabidopsis mutants with defects in systemic spreading of RNA interference (RNAi), or more specifically in root-to-shoot transmission of post-transcriptional gene silencing (rtp). To classify the mutants as either representing a new allele of a known or candidate gene versus carrying a mutation in an undiscovered gene, bulk genomic DNA from up to 23 independent mutants was used as template to amplify a collection of 47 known or candidate genes. These amplified sequences were combined into Can-Seq libraries and deep sequenced. Subsequently, mutations in the known and candidate genes were identified using a custom Snakemake script (https://github.com/Carroll-Lab/can_seq), and PCR zygosity tests were then designed and used to identify the individual mutants carrying each mutation. Using this approach, we showed that 28 of the 40 rtp mutants carried homozygous nonsense, missense or splice site mutations in one or more of the 47 known or candidate genes. We conducted complementation tests to demonstrate that several of the candidate mutations were responsible for the rtp defect. Importantly, by exclusion, the Can-Seq pipeline also identified rtp mutants that did not carry a causative mutation in any of the 47 known and candidate genes, and these mutants represent an undiscovered gene(s) required for systemic RNAi. CONCLUSIONS: Can-Seq offers an accurate, cost-effective method for classifying new mutants into known versus unknown genes. It has several advantages over existing genetic and DNA sequencing approaches that are currently being used in forward genetic screens for gene discovery. Using Can-Seq in conjunction with map-based gene cloning is a cost-effective approach towards identifying the full complement of genes contributing to a trait of interest.

The comparative analysis of the potential relationship between resveratrol and stilbene synthase gene family in the development stages of grapes (Vitis quinquangularis and Vitis vinifera).

Resveratrol is positively correlated with grapevine disease resistance and its consumption is also highly beneficial to human health. HPLC analyses showed that resveratrol content was significantly higher in most wild Chinese grapevines than in most European Vitis vinifera grapevine cvs. Fruit of the wild Chinese genotype Vitis quinquangularis Danfeng-2 contains much higher levels of resveratrol than some others. Because stilbene synthase is responsible for resveratrol biosynthesis, 41 full-length stilbene synthase genes were isolated from Danfeng-2 using the RACE method. A neighbor-joining tree of the STS family displayed high similarity between Danfeng-2 and V. vinifera cv. Pinot Noir. The content of the endogenous stilbene synthase family in tissues and the expression levels induced by powdery mildew were both higher in Danfeng-2 than in Pinot Noir. Moreover, expression in the berry was significantly higher than in the leaves. Our results demonstrated that resveratrol accumulation was consistent with endogenous STS gene expressions, and that both were higher in Danfeng-2 than in Pinot Noir. Therefore, STS genes and producing resveratrol from V. quinquangularis played more important role in Vitis resistance. Otherwise, the gene VqSTS6 was markedly higher than the other VqSTS genes in the six tissues/organs assayed by Real-time PCR, which will offer a useful basis for commercial application of resveratrol from Chinese wild grapes.

Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection.

Downy mildew, caused by the oomycete Plasmopara viticola, is a serious fungal disease in the cultivated European grapevines (Vitis vinifera L.). The class 10 of pathogenesis-related (PR) genes in grapevine leaves was reported to be accumulated at mRNA level in response to P. viticola infection. To elucidate the functional roles of PR10 genes during plant-pathogen interactions, a PR10 gene from a fungal-resistant accession of Chinese wild Vitis pseudoreticulata (designated VpPR10.2) was isolated and showed high homology to PR10.2 from susceptible V. vinifera (designated VvPR10.2). Comparative analysis displayed that there were significant differences in the patterns of gene expression between the PR10 genes from the two host species. VpPR10.2 was induced with high level in leaves infected by P. viticola, while VvPR10.2 showed a low response to this inoculation. Recombinant VpPR10.2 protein showed DNase activity against host genomic DNA and RNase activity against yeast total RNA in vitro. Meanwhile, recombinant VpPR10.2 protein inhibited the growth of tobacco fungus Alternaria alternata and over-expression of VpPR10.2 in susceptible V. vinifera enhanced the host resistance to P. viticola. The results from subcellular localization analysis showed that VpPR10.2 proteins were distributed dynamically inside or outside of host cell. Moreover, they were found in haustorium of P. viticola and nucleus of host cell which was associated with a nucleus collapse at 10 days post-inoculation. Taken together, these results suggested that VpPR10.2 might play an important role in host plant defense against P. viticola infection.

Three ERF transcription factors from Chinese wild grapevine Vitis pseudoreticulata participate in different biotic and abiotic stress-responsive pathways.

Ethylene response factor (ERF) functions as an important plant-specific transcription factor in regulating biotic and abiotic stress response through interaction with various stress pathways. We previously obtained three ERF members, VpERF1, VpERF2, and VpERF3 from a highly powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata cDNA full-length library. To explore their functions associated with plant disease resistance or biotic stress, we report here to characterize three ERF members from this library. PM-inoculation analysis on three different resistant grapevine genotypes revealed that three VpERFs displayed significant responses, but a different expression pattern. Over-expression of VpERF1, VpERF2, and VpERF3 in transgenic tobacco plants demonstrated that VpERF2 and VpERF3 enhanced resistance to both bacterial pathogen Ralstonia solanacearum and fungal pathogen Phytophtora parasitica var. nicotianae Tucker. Importantly, VpERF1-overexpressing transgenic Arabidopsis plants increased susceptibility toward these pathogens. Investigation on drought, cold, and heat treatments suggested, VpERF2 was distinctly induced, whereas VpERF3 displayed a very weak response and VpERF1 was distinctly induced by drought and heat. Concurrently, VpERF3 was significantly induced by salicylic acid (SA), methyl jasmonate (MeJA), and ET. Our results showed that the three VpERFs from Chinese wild V. pseudoreticulata play different roles in either preventing disease progression via regulating the expression of relevant defense genes, or directly involving abiotic stress responsive pathways.