Full genome sequence of a novel iflavirus from the aster leafhopper Macrosteles fascifrons.

The aster leafhopper Macrosteles fascifrons is a common insect pest that feeds on rice and other plants and may serve as a vector to transmit various viruses. Here, we discovered a novel virus from M. fascifrons using metagenomic sequencing. We obtained its complete genome sequence by contig assembly and rapid amplification of cDNA ends, and verified the genome sequence by Sanger sequencing of overlapping segments. Based on homology search and phylogenetic analysis, the new virus belongs to the family Iflaviridae and it is tentatively named "Macrosteles fascifrons iflavirus 1" (MfIV1). Excluding the poly(A) tail, the MfIV1 genome is 10,581 nucleotides in length and it is predicted to encode a polyprotein of 3119 amino acids long, which is likely further processed to several polypeptides with conserved domains, including two rhinovirus like (rhv-like) capsid domains, a cricket paralysis virus (CRPV) capsid domain, a helicase domain, and an RNA-dependent RNA polymerase (RdRp) domain. BLAST searches show that the highest amino acid sequence identity between the capsid proteins of MfIV1 and those of other reported iflaviruses is 60.22%, indicating that MfIV1 is a new member in the family Iflaviridae.

Roles of CsBRC1-like in leaf and lateral branch development in cucumber.

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes, as plant-specific transcription factors, play vital roles in flower pattern, leaf development and plant architecture. Our recent study shows that the TCP gene BRANCHED1 (CsBRC1) specifically regulates shoot branching in cucumber. Here, we found CsBRC1 had a closely related paralogous gene CsBRC1-like. The synteny analysis revealed that these two genes originated from a segmental duplication. CsBRC1-like displayed different expression patterns in cucumber compared with CsBRC1, indicating that they may have functional differentiation. Ectopic expression of CsBRC1-like in Arabidopsis brc1-1 mutant resulted in reduced rosette branches and rosette leaves, whereas silencing CsBRC1-like in cucumber only led to a deformed true leaf of seedling rather than affecting the shoot branching. RNA-seq analysis of wild-type and CsBRC1-like-RNAi plants implicated that CsBRC1-like might regulate early leaf development through affecting the transcripts of auxin and cytokinin related genes in cucumber. Moreover, CsBRC1-like directly interacts with CsTCP10a and CsBRC1 in vivo. Our results demonstrated that CsBRC1-like has a specific role in regulating leaf development, and CsBRC1-like and CsBRC1 may have overlapping roles in shoot branching.

PINOID is required for lateral organ morphogenesis and ovule development in cucumber.

Lateral organ development is essential for cucumber production. The protein kinase PINOID (PID) participates in distinct aspects of plant development by mediating polar auxin transport in different species. Here, we obtained a round leaf (rl) mutant that displayed extensive phenotypes including round leaf shape, inhibited tendril outgrowth, abnormal floral organs, and disrupted ovule genesis. MutMap+ analysis revealed that rl encodes a cucumber ortholog of PID (CsPID). A non-synonymous single nucleotide polymorphism in the second exon of CsPID resulted in an amino acid substitution from arginine to lysine in the rl mutant. Allelic testing using the mutant allele C356 with similar phenotypes verified that CsPID was the causal gene. CsPID was preferentially expressed in young leaf and flower buds and down-regulated in the rl mutant. Subcellular localization showed that the mutant form, Cspid, showed a dotted pattern of localization, in contrast to the continuous pattern of CsPID in the periphery of the cell and nucleus. Complementation analysis in Arabidopsis showed that CsPID, but not Cspid, can partially rescue the pid-14 mutant phenotype. Moreover, indole-3-acetic acid content was greatly reduced in the rl mutant. Transcriptome profiling revealed that transcription factors, ovule morphogenesis, and auxin transport-related genes were significantly down-regulated in the rl mutant. Biochemical analysis showed that CsPID physically interacted with a key polarity protein, CsREV (REVOLUTA). We developed a model in which CsPID regulates lateral organ morphogenesis and ovule development by stimulating genes related to auxin transport and ovule development.

CsBRC1 inhibits axillary bud outgrowth by directly repressing the auxin efflux carrier CsPIN3 in cucumber.

Shoot branching is an important agronomic trait that directly determines plant architecture and affects crop productivity. To promote crop yield and quality, axillary branches need to be manually removed during cucumber production for fresh market and thus are undesirable. Auxin is well known as the primary signal imposing for apical dominance and acts as a repressor for lateral bud outgrowth indirectly. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family gene BRANCHED1 (BRC1) has been shown to be the central integrator for multiple environmental and developmental factors that functions locally to inhibit shoot branching. However, the direct molecular link between auxin and BRC1 remains elusive. Here we find that cucumber BRANCHED1 (CsBRC1) is expressed in axillary buds and displays a higher expression level in cultivated cucumber than in its wild ancestor. Knockdown of CsBRC1 by RNAi leads to increased bud outgrowth and reduced auxin accumulation in buds. We further show that CsBRC1 directly binds to the auxin efflux carrier PIN-FORMED (CsPIN3) and negatively regulates its expression in vitro and in vivo. Elevated expression of CsPIN3 driven by the CsBRC1 promoter results in highly branched cucumber with decreased auxin levels in lateral buds. Therefore, our data suggest that CsBRC1 inhibits lateral bud outgrowth by direct suppression of CsPIN3 functioning and thus auxin accumulation in axillary buds in cucumber, providing a strategy to breed for cultivars with varying degrees of shoot branching grown in different cucumber production systems.

TMK1-mediated auxin signalling regulates differential growth of the apical hook.

The plant hormone auxin has crucial roles in almost all aspects of plant growth and development. Concentrations of auxin vary across different tissues, mediating distinct developmental outcomes and contributing to the functional diversity of auxin. However, the mechanisms that underlie these activities are poorly understood. Here we identify an auxin signalling mechanism, which acts in parallel to the canonical auxin pathway based on the transport inhibitor response1 (TIR1) and other auxin receptor F-box (AFB) family proteins (TIR1/AFB receptors)1,2, that translates levels of cellular auxin to mediate differential growth during apical-hook development. This signalling mechanism operates at the concave side of the apical hook, and involves auxin-mediated C-terminal cleavage of transmembrane kinase 1 (TMK1). The cytosolic and nucleus-translocated C terminus of TMK1 specifically interacts with and phosphorylates two non-canonical transcriptional repressors of the auxin or indole-3-acetic acid (Aux/IAA) family (IAA32 and IAA34), thereby regulating ARF transcription factors. In contrast to the degradation of Aux/IAA transcriptional repressors in the canonical pathway, the newly identified mechanism stabilizes the non-canonical IAA32 and IAA34 transcriptional repressors to regulate gene expression and ultimately inhibit growth. The auxin-TMK1 signalling pathway originates at the cell surface, is triggered by high levels of auxin and shares a partially overlapping set of transcription factors with the TIR1/AFB signalling pathway. This allows distinct interpretations of different concentrations of cellular auxin, and thus enables this versatile signalling molecule to mediate complex developmental outcomes.

LCM-seq reveals the crucial role of LsSOC1 in heat-promoted bolting of lettuce (Lactuca sativa L.).

Lettuce (Lactuca sativa L.) is one of the most economically important vegetables. The floral transition in lettuce is accelerated under high temperatures, which can significantly decrease yields. However, the molecular mechanism underlying the floral transition in lettuce is poorly known. Using laser capture microdissection coupled with RNA sequencing, we isolated shoot apical meristem cells from the bolting-sensitive lettuce line S39 at four critical stages of development. Subsequently, we screened specifically for the flowering-related gene LsSOC1 during the floral transition through comparative transcriptomic analysis. Molecular biology, developmental biology, and biochemical tools were combined to investigate the biological function of LsSOC1 in lettuce. LsSOC1 knockdown by RNA interference resulted in a significant delay in the timing of bolting and insensitivity to high temperature, which indicated that LsSOC1 functions as an activator during heat-promoted bolting in lettuce. We determined that two heat shock transcription factors, HsfA1e and HsfA4c, bound to the promoter of LsSOC1 to confirm that LsSOC1 played an important role in heat-promoted bolting. This study indicates that LsSOC1 plays a crucial role in the heat-promoted bolting process in lettuce. Further investigation of LsSOC1 may be useful for clarification of the bolting mechanism in lettuce.

Inflorescence Development and the Role of LsFT in Regulating Bolting in Lettuce (Lactuca sativa L.).

Lettuce (Lactuca sativa L.) is one of the most important leafy vegetable that is consumed during its vegetative growth. The transition from vegetative to reproductive growth is induced by high temperature, which has significant economic effect on lettuce production. However, the progression of floral transition and the molecular regulation of bolting are largely unknown. Here we morphologically characterized the inflorescence development and functionally analyzed the FLOWERING LOCUS T (LsFT) gene during bolting regulation in lettuce. We described the eight developmental stages during floral transition process. The expression of LsFT was negatively correlated with bolting in different lettuce varieties, and was promoted by heat treatment. Overexpression of LsFT could recover the late-flowering phenotype of ft-2 mutant. Knockdown of LsFT by RNA interference dramatically delayed bolting in lettuce, and failed to respond to high temperature. Therefore, this study dissects the process of inflorescence development and characterizes the role of LsFT in bolting regulation in lettuce.