Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing. In the current study, we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella, in addition to its known functions in the ovarian development in female adults and egg hatching. Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein (GFP) gene into autosomal Pxfl(2)d for labelling mutants could be achieved. However, this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity. We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9. The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences. We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein (CFP) expression cassettes on a Z-linked region in P. xylostella, which can also be used for early sex detection. By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain, the gene editing efficiency reached 29.73%, confirming the success of expressing a functional Cas9 gene. Taken together, we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P. xylostella, while the establishment of a heritable strain required the positioning of appropriate sites. This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

N6-methyladenosine (m6A) is one of the major epigenetic modifications in eukaryotes. Although increasing functions of m6A have been identified in insects, its role in Plutella xylostella L. for host plant adaptation remains unclear. In the current study, we show that the m6A content of P. xylostella was relatively low in different developmental stages and tissues, with no significant differences. Two RNA methyltransferase genes, PxMETTL3 (methyltransferase-like 3) and PxMETTL14 (methyltransferase-like 14), were identified and characterized. PxMETTL3 could be transcribed into two transcripts, and PxMETTL14 had only one transcript; both of these genes were highly expressed in egg and adult stages and reproductive tissues. The CRISPR/Cas9-mediated knockout of PxMETTL3 (ΔPxMETTL3-2) or PxMETTL14 (ΔPxMETTL14-14) confirmed their function in m6A installation into RNA. Furthermore, upon transfer from an artificial diet to the host plant, the mutant strains were affected in terms of larval and pupal weight or adult emergence rate, while the wildtype (WT) strain did not exhibit any difference. In addition, the fecundity and egg hatching rate of the WT strain decreased significantly, whereas only the ΔPxMETTL14-14 mutant strain displayed significantly decreased fecundity. There seemed to be a tradeoff between the stress adaptation and reproduction in P. xylostella mediated by m6A modification. During host transfer, the expression of PxMETTL14 was consistent with the change in m6A content, which implied that PxMETTL14 could respond to host plant defense effectively, and may regulate m6A content. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed transcripts with changes in m6A levels revealed that the potential functions of m6A-related genes may be involved in steroid biosynthesis for larval performance and metabolic pathways for adult reproduction. Overall, our work reveals an epigenetic regulation mechanism for the rapid adaptation of P. xylostella to variations in the host environment.

Glycoside hydrolase family 1 (GH1) members exhibit a broad substrate spectrum and play important roles in insect-plant interactions, such as the defensive β-glucosidase and β-thioglucosidase (so-called myrosinase). However, knowledge about the expression profiling and function of glycoside hydrolase family 1 members in a specialist pest of crucifers Plutella xylostella is still limited. In this study, 13 putative glycoside hydrolase family 1 members of P. xylostella were identified based on the sequence characteristics, while no myrosinase activity was detectable in P. xylostella using gas chromatography-mass spectrometry (GC-MS). Expression profiling of these glycoside hydrolase family 1 members identified the midgut-specific gene Px008848 that is induced by host plant. Further experiments revealed that the in vitro expressed Px008848 protein had β-glucosidase activity and the survival rate of the larvae feeding on wounded Arabidopsis thaliana leaves declined when leaves were treated with purified Px008848 protein. When CRISPR/Cas9-based homozygous mutant larvae of Px008848 and wild-type larvae were respectively transferred onto the A. thaliana, the larval survival rate of the mutant larvae was significantly higher than that of the wild-type individuals. Our work showed that certain insect glycoside hydrolase family 1 gene may have negative effect on the development of larvae feeding on the host plant, which broadened our understandings on the evolutionary function of this gene family in the insect-plant interaction.

[目的]RNA甲基化是基因转录后水平表观修饰的主要形式,参与了众多重要的细胞学过程.小菜蛾(Plutella xylostella)是危害十字花科蔬菜的重要寡食性害虫,与RNA甲基化相关基因的功能尚未见报道.本研究通过克隆小菜蛾的RNA甲基化蛋白同源基因fl(2)d,鉴定其表达模式,并敲除该基因以探究其生物学功能.[方法]通过小菜蛾基因组网站查找fl(2)d基因序列,PCR扩增其蛋白质编码序列(CDS);采用实时荧光定量PCR(qRT-PCR)技术,检测小菜蛾不同发育阶段个体以及成虫生殖腺中fl(2)d的相对表达量;运用CRISPR/Cas9结合卵的显微注射技术,对小菜蛾fl(2)d进行编辑;将fl(2)d被编辑过的成虫与野生型成虫杂交,并对其产生的后代进行近交,筛选fl(2)d突变品系;观测并比较突变体与野生型个体遗传特性、生物学参数和表型的差异,明确fl(2)d的功能.[结果]克隆得到长度为912 bp的fl(2)d CDS,fl(2)d在雌蛹、雌成虫和卵中的表达量较高,雄成虫和雄蛹的表达量较低,幼虫期的表达量最低,成虫卵巢中表达量显著高于精巢.通过向小菜蛾的卵注射靶向fl(2)d的向导RNA(sgRNA)和Cas9蛋白的混合物,对所产生的阳性后代进行10代的单对近交筛选,获得3种杂合的移码突变品系,分别缺失了4个(Δfl(2)d213-4)、5个(Δfl(2)d213-5)和7个(Δfl(2)d214-7)碱基.在上述品系的筛选过程中,发现了6只缺失4个碱基的纯合突变个体,2只缺失5个碱基的纯合突变个体;缺失4个碱基的纯合个体成功配成了两对,近交未产卵;剩余的2只缺失4个碱基的雄性纯合个体和2只缺失5个碱基的雄性纯合个体分别与同世代的雌性杂合突变个体近交后仍未产卵.说明fl(2)d纯合突变的个体存活率极低,且可能无法产生后代.通过分析后代基因型的分离比,发现杂合突变个体近交以及杂合突变个体与野生型个体杂交产生的后代中,杂合突变个体与野生型个体的比例分别略小于2和1,说明fl(2)d杂合突变会影响小菜蛾正常的生长发育,并导致部分个体死亡.杂合突变体后代中含有突变的雌雄个体比例接近1﹕1(P<0.05),推测小菜蛾fl(2)d可能与性别决定无关.只要是有突变品系小菜蛾所参与的交配,雌成虫产卵量和卵的孵化率均显著低于野生型(P<0.01),所产的卵多数发育异常,表现为失水皱缩、不能正常孵化.通过对成虫的生殖腺进行解剖,发现在野生型雌成虫与突变体雄成虫交配后,卵巢内卵的附着量较未交配的个体明显减少;未交配的突变体雌成虫卵巢内卵的附着量亦少于野生型,而突变体雄成虫的精巢未见明显异常.部分能够孵化的杂合突变个体在整个发育过程会发生不同程度的畸变,导致不能正常完成整个世代;另外一些杂合突变个体未见异常,可以将突变类型遗传给后代.根据上述发现,提出了基于fl(2)d的小菜蛾遗传防控模型.[结论]fl(2)d参与小菜蛾的生殖过程和胚胎发育,突变后显著影响后代种群数量,是开展小菜蛾遗传控制的理想靶标.

The TCP family genes have been under selection during domestication in maize and related andropogoneae crops. They encode plant-specific transcription factors involved in growth and development, especially in shaping the plant morphology and architecture. Sugarcane (Saccharum spp.) is the most productive in harvesting tonnage and 5th economically valuable crops worldwide for supporting world’s sugar and fuel ethanol production. Based on recently published sugarcane genome, we performed a genome-wide analysis of this gene family in the sugarcane genome and identified 22 TCP genes (SsTCPs), with 1–4 alleles each. They distributed across 28 chromosomes of S. spontaneum. Phylogenetic analysis showed that all 22 SsTCP genes can be classifed into two major groups: class I and class II. All 22 groups of SsTCPs showed species-specific clustering with TCPs of sorghum which indicate close relationship between sorghum and Saccharum. Structural organization of SsTCP genes showed that 37 SsTCPs are intronless and of the 22 SsTCPs with introns exist in coding region, which are different with TCPs of sorghum and wheat that located in UTR region. Expression study showed that transcripts of class I SsTCPs were more abundant than transcripts of class II SsTCPs. Moreover, the expression of SsTCP5–4, SsTCP6–2, SsTCP8–1, SsTCP12, SsTCP13, SsTCP15–1, SsTCP17–1 and SsTCP17–6 displayed significant change after plant hormones treatments, which suggest their function related to plant hormones. Cis-element analysis of SsTCPs’s promoter suggests that subfunctionalization may have occurred for homoeologous genes. Taken together, our analysis of TCPs in S. spontaneum provide a good starting for further studies to elucidate their specific function in sugarcane.

Plant microRNAs (miRNAs) have recently been reported to be involved in the cross-kingdom regulation of specific cellular and physiological processes in animals. However, little of this phenomenon is known for the communication between host plant and insect herbivore. In this study, the plant-derived miRNAs in the hemolymph of a cruciferous specialist Plutella xylostella were identified by small RNAs sequencing. A total of 39 miRNAs with typical characteristics of plant miRNAs were detected, of which 24 had read counts ≥ 2 in each library. Three plant-derived miRNAs with the highest read counts were validated, and all of them were predicted to target the hemocyanin domains-containing genes of P. xylostella. The luciferase assays in the Drosophila S2 cell demonstrated that miR159a and novel-7703-5p could target BJHSP1 and PPO2 respectively, possibly in an incomplete complementary pairing mode. We further found that treatment with agomir-7703-5p significantly influenced the pupal development and egg-hatching rate when reared on the artificial diet. The developments of both pupae and adults were severely affected upon their transfer to Arabidopsis thaliana, but this might be independent of the cross-kingdom regulation of the three plant-derived miRNAs on their target genes in P. xylostella, based on expression analysis. Taken together, our work reveals that the plant-derived miRNAs could break the barrier of the insect mid-gut to enter the circulatory system, and potentially regulate the development of P. xylostella. Our findings provide new insights into the co-evolution of insect herbivore and host plant, and novel direction for pest control using plant-derived miRNAs.