Biology subjects

Jakse, J.

Publications and source records attributed to Jakse, J..

5 recordsLinked to original sources

bioRxiv2026

Ommochrome pathway knockout via CRISPR/Cas9 reveals sex-linked eye pigmentation and establishes a heritable genome-editing platform in Rhynchophorus ferrugineus

The red palm weevil, Rhynchophorus ferrugineus, is the most economically destructive palm pest worldwide, threatening livelihoods, food security, and ecosystems across 49 countries. Weevil management currently relies predominantly on chemical insecticides, raising significant environmental and public health concerns. Despite its global agricultural importance, genetic approaches to pest management and the mechanistic basis of genome-editing strategies in Rhynchophorus remain largely unexplored. Here, we employed CRISPR/Cas9 genome editing to disrupt the R. ferrugineus ommochrome biosynthetic pathway -- a multi-enzymatic metabolic cascade that converts tryptophan into ommochrome pigments, including brown, yellow, and red pigments. We targeted two key pathway components: the ATP-binding cassette transporter white and the heme peroxidase cardinal. Both genes were ubiquitously expressed, with peak expression levels in the gut, fat body, and head. Elevated transcript levels were observed across early, mid, and late pupal stages and in 0-, 1-, and 2-day-old adult males and females, consistent with the progression of eye pigmentation throughout the R. ferrugineus life cycle. Embryonic microinjection of a single guide RNA (sgRNA)-Cas9 ribonucleoprotein complex targeting white produced in the Generation-0 (G0) adults with a distinct, white-eyed phenotype with a brownish outer margin, in contrast to the black eyes of wild-type adults. Genome-edited cardinal mutant adults displayed a translucent, brownish-white-eyed phenotype, with white streaks that gradually transitioned to a persistent translucent reddish-brown eye coloration. Mutations in both genes were confirmed in G0 adults by genomic DNA sequencing. Mutant adults were crossed to generate heterozygous G1 (+/-), G2 (-/-, -/+, and +/+), and G3 lines (-/-) with genotypes verified as carrying 2-, 3-, 9-, and 13-nucleotide deletions. A stable, heritable eye-color phenotype was established in homozygous knockout (-/-) G3 lines for both white and cardinal, confirmed by unambiguous indel (insertions/deletions) genotyping. Inheritance analysis revealed that both genes are X-linked, following a classical Mendelian sex-linked pattern: paternal alleles are transmitted exclusively to daughters, while maternal alleles are inherited equally by both daughters and sons. This study establishes the first fully homozygous knockout strain in R. ferrugineus and, by characterizing sex-linked inheritance in a coleopteran system, advances our understanding of how CRISPR/Cas9 can be efficiently applied to destructive palm weevil species. The present study represents the first report of CRISPR/Cas9 genome editing in any weevil (Curculionidae), using white and cardinal as marker genes. These findings provide a valuable platform for functional genomics and genome engineering in R. ferrugineus and offer a translational framework for genome editing in the invasive South American palm weevil, R. palmarum, laying a solid foundation for the development of gene-drive strategies aimed at sustainable palm weevil population control.

zoology↗

bioRxiv2023

Reverse chemical ecology approach for sustainable palm tree protection against invasive palm weevils

The reverse chemical ecology approach facilitates sustainable plant protection by identifying odorant receptors (ORs) tuned to odorants, especially the volatile molecules emitted from host plants that insects use for detection. A few studies have explored such an approach to develop sustainable pest management programs, especially in host-specialized insect species. We revealed the molecular mechanism of host plant detection of a destructive, invasive insect pest of palm trees (Arecaceae), the Asian palm weevil (Rhynchophorus ferrugineus), by deorphanizing an OR (RferOR2) tuned to several palm-emitted odors. We found that RferOR2 responded explicitly to several ecologically relevant palm-emitted odors and significantly to palm esters when transgenically expressed in Drosophila olfactory neurons. We mapped RferOR2 expression in the R. ferrugineus genome and found that odor specificity is likely to develop equally in both sexes. We inferred that the semiochemicals that attract palm weevils to a palm tree might aid in weevil control efforts by improving attraction, enticing many palm weevils to the traps. We demonstrate that including synthetic palm volatiles in pheromone-based mass trapping has a synergistic effect on pheromones, resulting in significantly increased weevil catches. We proved that insect OR deorphanization could aid in the identification of novel behaviorally active volatiles for inclusion in pest management. These results suggest that targeting RferOR2 may help design receptor antagonists that can interfere with weevil host-searching behavior in sustainable pest management applications. SignificanceAsian and South American palm weevils are tremendously important agricultural pests primarily adapted to palm trees and cause severe destruction, threatening sustainable palm cultivation worldwide. The host plant selection of these weevils is mainly attributed to functional specialization of odorant receptors that detect palm-derived volatiles. We unraveled the intricacies of weevil-palm tree communication by deorphanizing an odorant receptor tuned to natural palm-emitted odors. We used palm ester volatiles, which produced a significant response in the functional studies, and proved their synergistic effect on the pheromone coinciding with increased weevil catches in the field. We revealed that insect odorant receptor deorphanization could help identify novel behaviorally active volatiles (reverse chemical ecology) for sustainable palm protection.

ecology↗

bioRxiv2021

Plant genera Cannabis and Humulus share the same pair of well-differentiated sex chromosomes.

O_LIWe recently described, in Cannabis sativa, the oldest sex chromosome system documented so far in plants. Based on our estimate of its age, we predicted that it should be shared by its sister genus Humulus, which is known to also possess XY sex chromosomes. C_LIO_LIHere, we used transcriptome sequencing of a F1 family of Humulus lupulus to identify and study the sex chromosomes in this species using the probabilistic method SEX-DETector. C_LIO_LIWe identified 265 sex-linked genes in H. lupulus, located on the chromosome that is also the C. sativa sex chromosome pair. Using phylogenies of sex-linked genes, we show that a region of these chromosomes had already stopped recombining in the common ancestor of the two species. Furthermore, as in C. sativa, Y gene expression was reduced in correlation to the position on the X chromosome, and strongly Y degenerated genes showed dosage compensation. C_LIO_LIHere we report, for the first time in the Angiosperms, a sex chromosome system that is shared by two different genera. Recombination suppression started at least 21-25 My ago, and then (either gradually or step-wise) spread to a large part of the sex chromosomes, leading to a strongly degenerated Y. C_LI

evolutionary biology↗

bioRxiv2017

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion

BackgroundThe vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop.\n\nResultsThe secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed.\n\nConclusionsSeveral putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

microbiology↗

bioRxiv2017

Tomato immune receptor Ve1 recognizes surface-exposed co-localized N- and C-termini of Verticillium dahliae effector Ave1

Effectors are secreted by plant pathogens to facilitate infection, often through deregulation of host immune responses. During host colonization, race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae secrete the effector protein Ave1 that triggers immunity in tomato genotypes that encode the Ve1 immune receptor. Homologs of V. dahliae Ave1 (VdAve1) are found in plants and in few plant pathogenic microbes, and are differentially recognized by Ve1. However, how VdAve1 is recognized by Ve1 remained unknown. Interestingly, C-terminally affinity-tagged versions of VdAve1 failed to activate Ve1-mediated immunity, suggesting that exposure of the C-terminus of VdAve1 is required for Ve1-mediated recognition. This was confirmed by subsequent analysis of C-terminal deletion mutants, and by domain swap experiments. Although required, only the C-terminus of VdAve1 is not sufficient to activate Ve1-mediated immunity. Intriguingly, a three-dimensional structural model of VdAve1 revealed that the N- and C-termini co-localize on a surface-exposed patch of the VdAve1 protein. Indeed, subsequent analyses of N-terminal deletion mutants confirmed that also the N-terminus of VdAve1 is required to activate Ve1-mediated immunity. Thus, we conclude that a surface-exposed patch of the VdAve1 protein that is composed by co-localized N- and C-termini is recognized by the tomato immune receptor Ve1.

plant biology↗