Abstract Twenty-seven sets of simple sequence repeat (SSR) primers were developed through hybridizing of (CA)n, (CT)n and (GT)n and sequencing the positive clones in libraries constructed by using p50 silkworm strain. Of those primer pairs, 26 sets of SSR primers amplified well in two regional wild silkworm strains. Ten domesticated silkworm strains and two regional wild silkworm strains were used for comparing the polymorphisms and for constructing a phylogenetic tree employing the UPGAM method. The result showed that the genetic distances within Japanese strains are closer than those of Chinese strains. And this result also implies that Japanese strains diverged from domesticated silkworm later than Chinese strains. According to the clustering result, the domesticated silkworm is firstly clustered in one class, but could be classified into two groups. Within a strain, the individual polymorphism of wild silkworm was significantly higher in abundance than those of domesticated silkworm. The S SR primers of domesticated silkworm could be used in genetic studies for wild silkworm.
Abstract Insect double‐stranded (ds) RNA expression in transgenic crops can increase plant resistance to biotic stress; however, creating transgenic crops to defend against every insect pest is impractical. A rabidopsis Mob1A is required for organ growth and reproduction. When A rabidopsis roots were soaked in dsMob1A , the root lengths and numbers were significantly suppressed and plants could not bolt or flower. Twenty‐four hours after rice roots were immersed in fluorescent‐labelled dsEYFP (enhanced yellow fluorescent protein), fluorescence was observed in the rice sheath and stem and in planthoppers feeding on the rice. The expression levels of A go and D icer in rice and planthoppers were induced by dsEYFP . When rice roots were soaked in dsActin , their growth was also significantly suppressed. When planthoppers or A sian corn borers fed on rice or maize that had been irrigated with a solution containing the dsRNA of an insect target gene, the insect's mortality rate increased significantly. Our results demonstrate that dsRNAs can be absorbed by crop roots, trigger plant and insect RNAi and enhance piercing‐sucking and stem‐borer insect mortality rates. We also confirmed that dsRNA was stable under outdoor conditions. These results indicate that the root dsRNA soaking can be used as a bioinsecticide strategy during crop irrigation.
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.
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.
Summary 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 .
Intersimple sequence repeat (ISSR) amplification was used to analyze genetic relationships among silkworm, Bombyx mori L., strains. Nineteen primers containing simple sequence repeat (SSR) motifs were tested for amplification on a panel of 42 strains, representative of the diversity of silkworm germplasm; 12 of the primers amplified distinct, reproducible bands. The primers amplified a total of 108 bands, of which 85 (78.7%) were polymorphic. The ISSR results suggested that within the dinucleotide class, the poly(CA) motif was more common than the poly(CT) motif. The ISSR amplification pattern was used to group the silkworm strains into seven subclusters based on their origin in an unweighted pair-group method with arithmetic average cluster analysis by using Nei’s genetic distance. Seven major ecotypic silkworm groups were analyzed. Principal component analysis of the ISSR data supported the unweighted pair-group method with arithmetic average clustering. Therefore, ISSR amplification is a valuable method for determining genetic variability among silkworm varieties. This efficient genetic fingerprinting technique should be useful for characterizing the large numbers of silkworm strains held in national and international germplasm centers.
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.
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.
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