Periodic Reporting for period 4 - POLYADAPT (Molecular-genetic mechanisms of extreme adaptation in a polyphagous agricultural pest)
Período documentado: 2022-12-01 hasta 2023-11-30
Generalist (polyphagous) herbivores can feed and reproduce on many different plant species and include some of the most pesticide resistant and notorious pests in agriculture. An evolutionary link between host plant range and the development of pesticide resistance has been suggested. Although crucial for devising efficient crop protection strategies, the mechanisms underlying rapid adaptation are not well understood, especially in generalists. The spider mite Tetranychus urticae is a global pest known to feed on 1,100 different hosts from 140 plant families, including most major crops. With experimental advances and new tools developed for T. urticae, we are now poised for fundamental advances in understanding the molecular genetic make-up of adaption in generalist pests.
In POLYADAPT, we first generated a large collection of fully inbred and resistant mite strains and described the sampled genomic variation in the context of selection and adaptation, with a focus on gene copy number variation. We studied gene regulation mechanisms and quantified cis versus trans regulation of gene expression on a genome wide scale. Abundant trans-effects were identified related to detoxification gene families in multiple resistant strains. We then created a unique population resource via crossing schemes that allowed us to map master regulators of gene expression (an eQTL analysis). We found that a single trans eQTL hotspot controlled large differences in the expression of a subset of genes in different detoxification gene families, as well as other genes associated with host plant use. As validated by additional approaches, genetic variation in a nuclear hormone receptor related to HR96 was identified as the causal factor for gene expression variation.
Next, in several highly replicated experimental evolution studies, combined with Bulk Segregant Analysis (BSA), we uncovered without a prior hypothesis the genomic loci that underlie complex cases of resistance and plant adaptation. A core set of adaptation genes was validated by functional expression, electrophysiology and gene editing. For the latter, a revolutionary gene editing method was developed in POLYADPAT.
In summary, POLYADAPT has exploited and created new genomic tools that allowed to elucidate the molecular genetic mechanisms of extreme adaptation in polyphagous pests. This will in the long term lead to innovative methods of pest management.
In POLYADAPT, we first generated a large collection of fully inbred and resistant mite strains and described the sampled genomic variation in the context of selection and adaptation, with a focus on gene copy number variation. We studied gene regulation mechanisms and quantified cis versus trans regulation of gene expression on a genome wide scale. Abundant trans-effects were identified related to detoxification gene families in multiple resistant strains. We then created a unique population resource via crossing schemes that allowed us to map master regulators of gene expression (an eQTL analysis). We found that a single trans eQTL hotspot controlled large differences in the expression of a subset of genes in different detoxification gene families, as well as other genes associated with host plant use. As validated by additional approaches, genetic variation in a nuclear hormone receptor related to HR96 was identified as the causal factor for gene expression variation.
Next, in several highly replicated experimental evolution studies, combined with Bulk Segregant Analysis (BSA), we uncovered without a prior hypothesis the genomic loci that underlie complex cases of resistance and plant adaptation. A core set of adaptation genes was validated by functional expression, electrophysiology and gene editing. For the latter, a revolutionary gene editing method was developed in POLYADPAT.
In summary, POLYADAPT has exploited and created new genomic tools that allowed to elucidate the molecular genetic mechanisms of extreme adaptation in polyphagous pests. This will in the long term lead to innovative methods of pest management.
From a pool of field strains, inbred lines have been generated, phenotyped, sequenced and assembled.
Allele specific expression in F1 progeny of crosses has allowed to look at cis versus trans regulation of gene expression. A manuscript has been published.
Test crosses were performed in preparation of eQTL mapping. An eQTL experiment has been set up, more than 500 RNA samples were sequenced and data has been analysed. Master regulators of gene expression have been identified. The results have been published in Nature Communications.
A number of genetic mapping studies (experimental evolution + BSA) has been set up to unbiasedly map resistance loci in the genome. Multiple QTLs have been uncovered for a number of resistance cases. A number of QTL studies was published. A review outlining the method to assure easy access for other research groups has been published.
Pipelines for validation of resistance genes have been set up, mainly the E. coli and yeast expression systems, two electrode voltage clamp electrophysiology, spectrophotometric analysis of complex II electron flow.
Crispr/Cas9 transformation of spider mites has been achieved at the beginning of the project, but the method has been improved to reach unprecedented transformation efficiency. The method was recently published and a priority claim has been submitted to the European Patent Office.
Yeast-1Hybrid protocols have been designed, but the project switched to gene editing for validation of interactions.
Allele specific expression in F1 progeny of crosses has allowed to look at cis versus trans regulation of gene expression. A manuscript has been published.
Test crosses were performed in preparation of eQTL mapping. An eQTL experiment has been set up, more than 500 RNA samples were sequenced and data has been analysed. Master regulators of gene expression have been identified. The results have been published in Nature Communications.
A number of genetic mapping studies (experimental evolution + BSA) has been set up to unbiasedly map resistance loci in the genome. Multiple QTLs have been uncovered for a number of resistance cases. A number of QTL studies was published. A review outlining the method to assure easy access for other research groups has been published.
Pipelines for validation of resistance genes have been set up, mainly the E. coli and yeast expression systems, two electrode voltage clamp electrophysiology, spectrophotometric analysis of complex II electron flow.
Crispr/Cas9 transformation of spider mites has been achieved at the beginning of the project, but the method has been improved to reach unprecedented transformation efficiency. The method was recently published and a priority claim has been submitted to the European Patent Office.
Yeast-1Hybrid protocols have been designed, but the project switched to gene editing for validation of interactions.
We have unraveled a number of molecular-genetic mechanisms that allow generalist herbivores to rapidly adapt to new hosts and pesticides.
Using an allele specific design, we have quantified cis versus trans regulation of gene expression on a genome wide scale, revealing an important role of trans-regulation in detoxification gene families and other genes related to host plant use. This type of analysis has never been conducted on an economically important pest and examples in literature are limited to model organisms.
We then continued with an even more challenging experiment and mapped master regulators of gene expression in an eQTL analysis. This has not been conducted for any arthropod so far (except Drosophila) and generated a data-set that can be studied for the next 10 years. The experiment uncoverd a new family of transcription factors, that is expanded in T. urticae and lies at the basis of its strong transcriptional response and adaptation potential.
Inbred lines were also used in an experimental evolution setup combined with Bulk Segregant Analysis (BSA) to map the genomic loci that underlie both simple and complex cases of resistance and plant adaptation. This forward genetic approach is unique in the field and has defined adaptation loci regardless of prior hypotheses (functional coding variants, regulation variants, etc.).
Finally and importantly, validation of the results in functional assays has been a prime focus. Of very high importance, we succeeded in devising a gene-editing protocol for this (and other difficult to transform) species, called SYNCAS, that will benefit a very large scientific community.
Using an allele specific design, we have quantified cis versus trans regulation of gene expression on a genome wide scale, revealing an important role of trans-regulation in detoxification gene families and other genes related to host plant use. This type of analysis has never been conducted on an economically important pest and examples in literature are limited to model organisms.
We then continued with an even more challenging experiment and mapped master regulators of gene expression in an eQTL analysis. This has not been conducted for any arthropod so far (except Drosophila) and generated a data-set that can be studied for the next 10 years. The experiment uncoverd a new family of transcription factors, that is expanded in T. urticae and lies at the basis of its strong transcriptional response and adaptation potential.
Inbred lines were also used in an experimental evolution setup combined with Bulk Segregant Analysis (BSA) to map the genomic loci that underlie both simple and complex cases of resistance and plant adaptation. This forward genetic approach is unique in the field and has defined adaptation loci regardless of prior hypotheses (functional coding variants, regulation variants, etc.).
Finally and importantly, validation of the results in functional assays has been a prime focus. Of very high importance, we succeeded in devising a gene-editing protocol for this (and other difficult to transform) species, called SYNCAS, that will benefit a very large scientific community.