Research Article

Horizontal gene transfer allowed the emergence of broad host range entomopathogens

Qiangqiang Zhang, Xiaoxuan Chen, Chuan Xu, Hong Zhao, Xing Zhang, Guohong Zeng, Ying Qian, Ran Liu, Na Guo, Wubin Mi, Yamin Meng, Raymond J. St. Leger, and Weiguo Fang
PNAS April 16, 2019 116 (16) 7982-7989; first published April 4, 2019; https://doi.org/10.1073/pnas.1816430116

  1. Edited by Thomas A. Richards, University of Exeter, Exeter, United Kingdom, and accepted by Editorial Board Member W. F. Doolittle March 10, 2019 (received for review September 24, 2018)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    Distribution of homologs of the M. robertsii HGT genes in other Metarhizium species. (Left) A reconstruction of the phylogeny of seven Metarhizium species based on genomic sequences (n = 7). Pink branches indicate specialists; green branches indicate species with intermediate host range; red branches indicate generalists. (Right) The presence of homologs of the HGT genes found in M. robertsii in other Metarhizium species. Black box, present; white box, absent. Note: homologs of the 13 HGT candidates found in M. robertsii were absent in the specialist M. album, 7 of which were also absent in another specialist, M. acridum.

  • Fig. 2.
    Fig. 2.

    Expression and regulation of HGT genes in M. robertsii. (A) qRT-PCR analysis of gene expression during saprophytic growth (grown in nutrient-rich SDY medium), two key infection stages (cuticle, cuticle penetration; hemolymph, hemocoel colonization), and plant root colonization (rhizosphere). The expression level of a gene during saprophytic growth (i.e., in SDY) is set to 1; the values represent the log2-transformed fold changes of differential gene expression during infection or root colonization compared with the saprophytic growth in SDY. (B) qRT-PCR analysis of expression of the 12 HGT genes up-regulated during cuticle penetration (shown in A) in WT and 6 signaling mutants (impaired in cuticle penetration). These include three MAPK mutants (ΔMero-Fus3, ΔMero-Hog1, and ΔMero-Slt2), the PKA mutant ΔMaPKA, the membrane protein Mr-OPY2 mutant ΔMr-OPY2, and the adaptor Mr-STE50 mutant ΔMr-STE50. The expression level of WT is set to 1; the values represent the log2-transformed fold changes of differential gene expression of mutants vs. WT. The experiments were repeated three times, and the three individual experiments are indicated by numbers 1–3, respectively.

  • Fig. 3.
    Fig. 3.

    Ligand binding of two HGT-derived lipid carriers (MAA_05244 and MAA_05652). Binding of recombinant MAA_05652 (Left) and MAA_05244 (Right) to hydrocarbons and fatty acids. All assays were repeated three times. Data are expressed as mean ± SE.

  • Fig. 4.
    Fig. 4.

    The impact of deleting HGT protease genes on the ability of M. robertsii to degrade the insect cuticle and produce cuticle-degrading enzymes. WT, the three single gene-deletion mutants (ΔMAA_00986, ΔMAA_01413, ΔMAA_09637), and the triple gene-deletion mutant (ΔMAA_00986׃׃ΔMAA_01413׃׃ΔMAA_09637) were grown on locust cuticle as the sole carbon and nitrogen source. (A) The concentrations of amino acids (Left) and peptides (Right) in culture supernatants. Controls were uninoculated insect cuticle medium. (B) Pr1 protease activity (Left), total extracellular protease activity (Middle), and chitinase activity (Right) in culture supernatants. All assays were repeated three times. Data are expressed as mean ± SE. Values with different letters are significantly different (n = 3; P < 0.05, Tukey’s test in one-way ANOVA).

  • Fig. 5.
    Fig. 5.

    Heterologous expression of five M. robertsii HGT genes expanded the host range of the specialist M. acridum. (A) Survival curves of G. mellonella larvae (a nonnatural host of M. acridum) infected by WT M. acridum and M. acridum transformants expressing the acetyltransferase MAA_00129, the protease MAA_01413, or three hypothetic proteins (MAA_08604, MAA_08605, or MAA_08684). Note: WT M. acridum does not infect G. mellonella, whereas transformants cause approximately 10–20% mortality. All assays were repeated three times. Data are expressed as mean ± SE. Note: no significant differences in virulence were found between transformants. (B) Amino acid (Left) and peptide (Right) concentrations in culture supernatants of WT M. acridum and transgenic M. acridum (T-MAA_01413) expressing the protease MAA_01413. Cultures were grown on G. mellonella (nonnatural host) cuticle as sole source of carbon and nitrogen. Controls were uninoculated insect cuticle medium. (C) Pr1 protease activity (Left), total extracellular protease activity (Middle), and chitinase activity (Right) in the culture supernatants described in B. All assays were repeated three times. Data are expressed as mean ± SE. Values with different letters are significantly different (n = 3; P < 0.05, Tukey’s test in one-way ANOVA).

Tables

  • Table 1.

    Virulence (expressed as LT50 values) of WT and HGT gene mutants

    StrainsVirulence, LT50
    Topical applicationInjectionGenes deleted or knocked down
    WT9.1 ± 0.57*4.8 ± 0.35*—
    Single gene-deletion mutants
     ΔMAA_0141315.2 ± 1.87†5.2 ± 0.35*Protease
     ΔMAA_0098615.3 ± 1.51†5.1 ± 0.18*Protease
     ΔMAA_0963716.7 ± 1.27†5.3 ± 0.19*Pyroglutamyl peptidase
     ΔMAA_052449.3 ± 0.28*4.8 ± 0.26*Lipid carrier
     ΔMAA_0565213.9 ± 1.07†5.2 ± 0.12*Lipid carrier
     ΔMAA_0860512.5 ± 0.93†4.9 ± 0.38*Hypothetical protein
    Multiple gene-deletion mutants
     ΔMAA_00986::ΔMAA_09637::ΔMAA_0141323.7 ± 3.28‡4.5 ± 0.13*Three proteases
     ΔMAA_00986::ΔMAA_09637::ΔMAA_01413::MAA_05652kd #1NC (8.8%)4.8 ± 0.14*Three proteases, one lipid carrier
     ΔMAA_05244::ΔMAA_0565222.2 ± 0.91‡4.0 ± 0.15*Two lipid carriers
     ΔMAA_05244::ΔMAA_05652::ΔMAA_03817NC (12.8%)5.9 ± 0.13†Three lipid carriers
     ΔMAA_05244::ΔMAA_05652::ΔMAA_03817::MAA_00986kd #1NC (0.9%)6.1 ± 0.12†Three lipid carriers, one protease

    • kd, knockdown; LT50, time required to kill 50% of the insects, NC, not calculated because of low mortality rate (rate in parentheses). This table shows only the LT50s of mutants disrupted in multiple genes and single gene-deletion (Δ) mutants significantly different from WT following topical application. Data on other mutants, including complemented deletion mutants, are shown in SI Appendix, Table S3. For each inoculation method (i.e., each column), values followed by different symbols (*, †, ‡) are significantly different (P < 0.05, Tukey’s test in one-way ANOVA). All assays were repeated three times. Data are expressed as the mean ± SE.

Data supplements

Back to top
Article Alerts
Email Article
Citation Tools
Request Permissions
Horizontal gene transfer allowed the emergence of broad host range entomopathogens
Qiangqiang Zhang, Xiaoxuan Chen, Chuan Xu, Hong Zhao, Xing Zhang, Guohong Zeng, Ying Qian, Ran Liu, Na Guo, Wubin Mi, Yamin Meng, Raymond J. St. Leger, Weiguo Fang
Proceedings of the National Academy of Sciences Apr 2019, 116 (16) 7982-7989; DOI: 10.1073/pnas.1816430116
Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo
Proceedings of the National Academy of Sciences: 116 (16)
Table of Contents

Submit

Article Classifications

Jump to section

You May Also be Interested in

Similar Articles