Conserved transcription factors NRZ1 and NRM1 regulate NLR receptor-mediated immunity.

Plant innate immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors plays an important role in defense against various pathogens. Although key biochemical events involving NLR activation and signaling have been recently uncovered, we know very little about the transcriptional regulation of NLRs and their downstream signaling components. Here, we show that the Toll-Interleukin 1 receptor homology domain containing NLR (TNL) gene N (Necrosis), which confers resistance to Tobacco mosaic virus, is transcriptionally induced upon immune activation. We identified two conserved transcription factors, N required C3H zinc finger 1 (NRZ1) and N required MYB-like transcription factor 1 (NRM1), that activate N in an immune responsive manner. Genetic analyses indicated that NRZ1 and NRM1 positively regulate coiled-coil domain-containing NLR- and TNL-mediated immunity and function independently of the signaling component Enhanced Disease Susceptibility 1. Furthermore, NRZ1 functions upstream of NRM1 in cell death signaling, and their gene overexpression induces ectopic cell death and expression of NLR signaling components. Our findings uncovered a conserved transcriptional regulatory network that is central to NLR-mediated cell death and immune signaling in plants.

Dicer-like2b suppresses the wiry leaf phenotype in tomato induced by tobacco mosaic virus.

Dicer-like (DCL) proteins are principal components of RNA silencing, a major defense mechanism against plant virus infections. However, their functions in suppressing virus-induced disease phenotypes remain largely unknown. Here, we identified a role for tomato (Solanum lycopersicum) DCL2b in regulating the wiry leaf phenotype during defense against tobacco mosaic virus (TMV). Knocking out SlyDCL2b promoted TMV accumulation in the leaf primordium, resulting in a wiry phenotype in distal leaves. Biochemical and bioinformatics analyses showed that 22-nt virus-derived small interfering RNAs (vsiRNAs) accumulated less abundantly in slydcl2b mutants than in wild-type plants, suggesting that SlyDCL2b-dependent 22-nt vsiRNAs are required to exclude virus from leaf primordia. Moreover, the wiry leaf phenotype was accompanied by upregulation of Auxin Response Factors (ARFs), resulting from a reduction in trans-acting siRNAs targeting ARFs (tasiARFs) in TMV-infected slydcl2b mutants. Loss of tasiARF production in the slydcl2b mutant was in turn caused by inhibition of miRNA390b function. Importantly, silencing SlyARF3 and SlyARF4 largely restored the wiry phenotype in TMV-infected slydcl2b mutants. Our work exemplifies the complex relationship between RNA viruses and the endogenous RNA silencing machinery, whereby SlyDCL2b protects the normal development of newly emerging organs by excluding virus from these regions and thus maintaining developmental silencing.

Identification of CaPCR1, an OFP gene likely involved in pointed versus concave fruit tip regulation in pepper (Capsicum annuum L.) using recombinant inbred lines.

KEY MESSAGE: CaPCR1 (Capana12g002165) was a candidate gene regulating fruit concave/pointed tip shape in pepper. The concave shape of the fruit tip in pepper plants is highly susceptible to drought and low temperature stresses, resulting in the appearance of a pointed tip fruit, which affects its commercial value. However, few studies on the process of fruit tip development and regulatory genes in pepper have been reported. Herein, the developmental process of the ovary before anthesis, especially changes in the shape of the ovary tip, was studied in detail. The results showed that the final fruit tip shape was consistent with the ovary tip shape before anthesis, and a concave tip shape gradually developed. F4 recombinant inbred lines (RILs) were constructed to map the genes regulating fruit tip shape through hybridization of the LRS and SBS pepper inbred lines. CaPCR1 (Capana12g002165), an OFP (OVATE Family Protein) family gene, was located in the candidate region on chr12. Three SNPs were found in the protein coding sequence of CaPCR1 between SBS and LRS, but only one SNP led to amino acid variation. Sequence variations, including base replacements, deletions and insertions, were also detected in the gene promoter region. The relative expression level of the CaPCR1 gene was significantly greater in the concave tip ovary than in the pointed tip ovary. qRT‒PCR analysis revealed that the CaPCR1 gene was expressed mainly in the gynoecium, placenta and green fruit pericarp, which was consistent with its function in ovary and fruit development. Taken together, these results suggested that CaPCR1 is a candidate gene involved in fruit tip shape determination in pepper.

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made toward understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57 862 high-quality full-length mRNA sequences derived from 18 362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://bigd.big.ac.cn/gsa Accession number, CRA001412.

A role for small RNA in regulating innate immunity during plant growth.

Plant genomes encode large numbers of nucleotide-binding (NB) leucine-rich repeat (LRR) immune receptors (NLR) that mediate effector triggered immunity (ETI) and play key roles in protecting crops from diseases caused by devastating pathogens. Fitness costs are associated with plant NLR genes and regulation of NLR genes by micro(mi)RNAs and phased small interfering RNAs (phasiRNA) is proposed as a mechanism for reducing these fitness costs. However, whether NLR expression and NLR-mediated immunity are regulated during plant growth is unclear. We conducted genome-wide transcriptome analysis and showed that NLR expression gradually increased while expression of their regulatory small RNAs (sRNA) gradually decreased as plants matured, indicating that sRNAs could play a role in regulating NLR expression during plant growth. We further tested the role of miRNA in the growth regulation of NLRs using the tobacco mosaic virus (TMV) resistance gene N, which was targeted by miR6019 and miR6020. We showed that N-mediated resistance to TMV effectively restricted this virus to the infected leaves of 6-week old plants, whereas TMV infection was lethal in 1- and 3-week old seedlings due to virus-induced systemic necrosis. We further found that N transcript levels gradually increased while miR6019 levels gradually decreased during seedling maturation that occurs in the weeks after germination. Analyses of reporter genes in transgenic plants showed that growth regulation of N expression was post-transcriptionally mediated by MIR6019/6020 whereas MIR6019/6020 was regulated at the transcriptional level during plant growth. TMV infection of MIR6019/6020 transgenic plants indicated a key role for miR6019-triggered phasiRNA production for regulation of N-mediated immunity. Together our results demonstrate a mechanistic role for miRNAs in regulating innate immunity during plant growth.

High-quality assembled and annotated genomes of Nicotiana tabacum and Nicotiana benthamiana reveal chromosome evolution and changes in defense arsenals.

Nicotiana tabacum and Nicotiana benthamiana are widely used models in plant biology research. However, genomic studies of these species have lagged. Here we report the chromosome-level reference genome assemblies for N. benthamiana and N. tabacum with an estimated 99.5% and 99.8% completeness, respectively. Sensitive transcription start and termination site sequencing methods were developed and used for accurate gene annotation in N. tabacum. Comparative analyses revealed evidence for the parental origins and chromosome structural changes, leading to hybrid genome formation of each species. Interestingly, the antiviral silencing genes RDR1, RDR6, DCL2, DCL3, and AGO2 were lost from one or both subgenomes in N. benthamiana, while both homeologs were kept in N. tabacum. Furthermore, the N. benthamiana genome encodes fewer immune receptors and signaling components than that of N. tabacum. These findings uncover possible reasons underlying the hypersusceptible nature of N. benthamiana. We developed the user-friendly Nicomics (http://lifenglab.hzau.edu.cn/Nicomics/) web server to facilitate better use of Nicotiana genomic resources as well as gene structure and expression analyses.

The role of transposon inverted repeats in balancing drought tolerance and yield-related traits in maize.

The genomic basis underlying the selection for environmental adaptation and yield-related traits in maize remains poorly understood. Here we carried out genome-wide profiling of the small RNA (sRNA) transcriptome (sRNAome) and transcriptome landscapes of a global maize diversity panel under dry and wet conditions and uncover dozens of environment-specific regulatory hotspots. Transgenic and molecular studies of Drought-Related Environment-specific Super eQTL Hotspot on chromosome 8 (DRESH8) and ZmMYBR38, a target of DRESH8-derived small interfering RNAs, revealed a transposable element-mediated inverted repeats (TE-IR)-derived sRNA- and gene-regulatory network that balances plant drought tolerance with yield-related traits. A genome-wide scan revealed that TE-IRs associate with drought response and yield-related traits that were positively selected and expanded during maize domestication. These results indicate that TE-IR-mediated posttranscriptional regulation is a key molecular mechanism underlying the tradeoff between crop environmental adaptation and yield-related traits, providing potential genomic targets for the breeding of crops with greater stress tolerance but uncompromised yield.

A Conserved, Serine-Rich Protein Plays Opposite Roles in N-Mediated Immunity against TMV and N-Triggered Cell Death.

Plant nucleotide-binding, leucine-rich, repeat-containing proteins (NLRs) play important roles in plant immunity. NLR expression and function are tightly regulated by multiple mechanisms. In this study, a conserved serine/arginine-rich protein (SR protein) was identified through the yeast one-hybrid screening of a tobacco cDNA library using DNA fragments from the N gene, an NLR that confers immunity to tobacco mosaic virus (TMV). This SR protein showed an interaction with a 3' genomic regulatory sequence (GRS) and has a potential role in regulating the alternative splicing of N. Thus, it was named SR regulator for N, abbreviated SR4N. Further study showed that SR4N plays a positive role in N-mediated cell death but a negative role in N protein accumulation. SR4N also promotes multiple virus replications in co-expression experiments, and this enhancement may not function through RNA silencing suppression, as it did not enhance 35S-GFP expression in co-infiltration experiments. Bioinformatic and molecular studies revealed that SR4N belongs to the SR2Z subtype of the SR protein family, which was conserved in both dicots and monocots, and its roles in repressing viral immunity and triggering cell death were also conserved. Our study revealed new roles for SR2Z family proteins in plant immunity against viruses.

A New Biotin Labeling and High-Molecular-Weight RNA Northern Method and Its Application in Viral RNA Detection.

Viruses cause severe crop losses. Studying the interaction between viruses and plants is very important for development of control measures. Northern blot is a well-accepted but very challenging technique to monitor the infection of viruses. Here, we modified the high-molecular-weight (hmw)RNA Northern blot experiment process, utilizing vertical electrophoresis to separate the RNA with denatured agarose gel. This protocol is compatible with regular equipment for Western blots and small RNA Northern blots and requires less input of total RNA. A new method to label the probe with biotin was also developed, which requires commonly used T4 DNA polymerase and detects viral RNA with high sensitivity. These new protocols made hmwRNA Northern blot cost-effective and easy-to-operate, very suitable for studying virus-host interactions.

[Effect of basic fibroblast growth factor on wound healing of bilioenteric anastomosis].

OBJECTIVE: To observe the effects of basic fibroblast growth factor (bFGF) on wound healing of bilioenteric anastomosis as to prevent postoperative biliary strictures. METHODS: A model of choledochoduodenostomy in 31 dogs were constructed at first, then bFGF was administered on the local anastomosis (bFGF group) for 1 week after the operation as compared with sodium chloride solution (control group). Both groups were observed with light microscope (HE, Masson staining) and electron microscope 3 days, 1 and 3 weeks and 3 and 6 months after the operation (n=3). Hydroxyproline was measured at the same time. RESULTS: bFGF group healed more quickly compared with control group. Good function of cells were detected by electron microscope. Better mucosa epithelia, fibroblast, and capillary vessel proliferation were detected by histological observation in bFGF group. In bFGF group, collagen fibers were arranged orderly. Three weeks after operation, collagen fibers in control group were orderless and in whirlpool. Hydroxyproline of bFGF group was lower than that of control group (P<0.05). CONCLUSION: bFGF administered in local anastomosis is an effective method to prevent postoperative anastomotic stenosis.