Enhanced UV Resistance and Improved Killing of Malaria Mosquitoes by Photolyase Transgenic Entomopathogenic Fungi

<div><p>The low survival of microbial pest control agents exposed to UV is the major environmental factor limiting their effectiveness. Using gene disruption we demonstrated that the insect pathogenic fungus <em>Metarhizium robertsii</em> uses photolyases to remove UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) photoproducts [(6-4)PPs] from its DNA. However, this photorepair is insufficient to fix CPD lesions and prevent the loss of viability caused by seven hours of solar radiation. Expression of a highly efficient archaeal (<em>Halobacterium salinarum</em>) CPD photolyase increased photorepair >30-fold in both <em>M. robertsii</em> and <em>Beauveria bassiana</em>. Consequently, transgenic strains were much more resistant to sunlight and retained virulence against the malaria vector <em>Anopheles gambiae</em>. In the field this will translate into much more efficient pest control over a longer time period. Conversely, our data shows that deleting native photolyase genes will strictly contain <em>M. robertsii</em> to areas protected from sunlight, alleviating safety concerns that transgenic hypervirulent <em>Metarhizium</em> spp will spread from mosquito traps or houses. The precision and malleability of the native and transgenic photolyases allows design of multiple pathogens with different strategies based on the environments in which they will be used.</p> </div

The effectiveness of the <i>Metarhizium</i> transcription factor (maa08770) nuclear localization signal peptide (NLS) at directing green fluorescent protein (GFP) into fungal nuclei.

<p>Upper row: <i>M. robertsii</i>. Lower row: <i>B. bassiana</i>. DIC: Differential interference contrast; DAPI: nuclear DNA was stained with DAPI (blue, 4′,6′-diamidino-2-phenylindole); GFP: green fluorescent signal in nuclei; n: nucleus.</p

Germination rates of spores of wild type <i>B. bassisana</i> and the transgenic strain Bb-HsPHR2 radiated by UV (followed by either photoreactivation or NER) and by sunlight.

<p>Germination rates of spores of wild type <i>B. bassisana</i> and the transgenic strain Bb-HsPHR2 radiated by UV (followed by either photoreactivation or NER) and by sunlight.</p

Germination rates of spores of wild type and transgenic <i>M. robertsii</i> radiated by UV (followed by either photoreactivation or NER) and by sunlight.

<p>Germination rates of spores of wild type and transgenic <i>M. robertsii</i> radiated by UV (followed by either photoreactivation or NER) and by sunlight.</p

Kinetics of mosquito survivorship in bioassays.

<p>Adult female mosquitoes (<i>A. gambiae</i>) were sprayed with <i>M. robertsii</i> spore suspensions (1×10⁷ spores/mL) that had been irradiated by sunlight for 7 h. Red = wild type <i>M. robertsii</i>; Blue = <i>M. robertsii</i> expressing a CPD photolyase (HsPHR2) from <i>H. salinarum</i>; black = control insects that were treated with 0.05% Tween-80.</p

Quantification of CPDs in solar-irradiated mycelia with ELISA.

<p>Upper panel: the amount of CPDs in wild type and transgenic <i>M. robertsii</i> expressing a CPD photolyase (HsPHR2). Down panel: a representative ELISA assay plate using CPD monoclonal antibody. WT: wild type; TS: a transgenic strain expressing HsPHR2.</p

Germination rates of spores of <i>ΔMrphr1, ΔMrphr2, ΔMrphr1ΔMrphr2</i> and the wild type irradiated by UV followed by either photoreactivation or NER.

<p>Germination rates of spores of <i>ΔMrphr1, ΔMrphr2, ΔMrphr1ΔMrphr2</i> and the wild type irradiated by UV followed by either photoreactivation or NER.</p

The genetic basis for variation in resistance to infection in the <i>Drosophila melanogaster</i> genetic reference panel

<div><p>Individuals vary extensively in the way they respond to disease but the genetic basis of this variation is not fully understood. We found substantial individual variation in resistance and tolerance to the fungal pathogen <i>Metarhizium anisopliae</i> Ma549 using the <i>Drosophila melanogaster</i> Genetic Reference Panel (DGRP). In addition, we found that host defense to Ma549 was correlated with defense to the bacterium <i>Pseudomonas aeruginosa</i> Pa14, and several previously published DGRP phenotypes including oxidative stress sensitivity, starvation stress resistance, hemolymph glucose levels, and sleep indices. We identified polymorphisms associated with differences between lines in both their mean survival times and microenvironmental plasticity, suggesting that lines differ in their ability to adapt to variable pathogen exposures. The majority of polymorphisms increasing resistance to Ma549 were sex biased, located in non-coding regions, had moderately large effect and were rare, suggesting that there is a general cost to defense. Nevertheless, host defense was not negatively correlated with overall longevity and fecundity. In contrast to Ma549, minor alleles were concentrated in the most Pa14-susceptible as well as the most Pa14-resistant lines. A pathway based analysis revealed a network of Pa14 and Ma549-resistance genes that are functionally connected through processes that encompass phagocytosis and engulfment, cell mobility, intermediary metabolism, protein phosphorylation, axon guidance, response to DNA damage, and drug metabolism. Functional testing with insertional mutagenesis lines indicates that 12/13 candidate genes tested influence susceptibility to Ma549. Many candidate genes have homologs identified in studies of human disease, suggesting that genes affecting variation in susceptibility are conserved across species.</p></div

Cellular networks of candidate genes.

<p>Enriched cellular genetic pathway for candidate genes from all genome wide association analyses (squares), allowing one missing gene (white triangles) or compound (white circles). The border colors indicate the over-represented gene ontology categories (<i>P</i><0.005): axon guidance (red), translation initiation (orange), protein phosphorylation (dark green), cell migration (magenta), phagocytosis and engulfment (yellow), arginine and proline metabolism (dark blue), purine metabolism (light blue), response to DNA damage (light green), ether lipid metabolism (gray), glycerolipid metabolism (teal), P450 related drug metabolism (brown).</p

Time course of CFU production in male flies from DGRP lines.

<p>Flies were homogenized and plated at 12 h intervals post-infection until death. Ten lines are shown as representative examples. CFUs were averaged from ten individual flies per fly line per time point.</p