Image2_Insights into the differences related to the resistance mechanisms to the highly toxic fruit Hippomane mancinella (Malpighiales: Euphorbiaceae) between the larvae of the sister species Anastrepha acris and Anastrepha ludens (Diptera: Tephritidae) through comparative transcriptomics.pdf

The Manchineel, Hippomane mancinella (“Death Apple Tree”) is one of the most toxic fruits worldwide and nevertheless is the host plant of the monophagous fruit fly species Anastrepha acris (Diptera: Tephritidae). Here we aimed at elucidating the detoxification mechanisms in larvae of A. acris reared on a diet enriched with the toxic fruit (6% lyophilizate) through comparative transcriptomics. We compared the performance of A. acris larvae with that of the sister species A. ludens, a highly polyphagous pest species that is unable to infest H. mancinella in nature. The transcriptional alterations in A. ludens were significantly greater than in A. acris. We mainly found two resistance mechanisms in both species: structural, activating cuticle protein biosynthesis (chitin-binding proteins likely reducing permeability to toxic compounds in the intestine), and metabolic, triggering biosynthesis of serine proteases and xenobiotic metabolism activation by glutathione-S-transferases and cytochrome P450 oxidoreductase. Some cuticle proteins and serine proteases were not orthologous between both species, suggesting that in A. acris, a structural resistance mechanism has been selected allowing specialization to the highly toxic host plant. Our results represent a nice example of how two phylogenetically close species diverged over recent evolutionary time related to resistance mechanisms to plant secondary metabolites.</p

Image1_Insights into the differences related to the resistance mechanisms to the highly toxic fruit Hippomane mancinella (Malpighiales: Euphorbiaceae) between the larvae of the sister species Anastrepha acris and Anastrepha ludens (Diptera: Tephritidae) through comparative transcriptomics.pdf

The Manchineel, Hippomane mancinella (“Death Apple Tree”) is one of the most toxic fruits worldwide and nevertheless is the host plant of the monophagous fruit fly species Anastrepha acris (Diptera: Tephritidae). Here we aimed at elucidating the detoxification mechanisms in larvae of A. acris reared on a diet enriched with the toxic fruit (6% lyophilizate) through comparative transcriptomics. We compared the performance of A. acris larvae with that of the sister species A. ludens, a highly polyphagous pest species that is unable to infest H. mancinella in nature. The transcriptional alterations in A. ludens were significantly greater than in A. acris. We mainly found two resistance mechanisms in both species: structural, activating cuticle protein biosynthesis (chitin-binding proteins likely reducing permeability to toxic compounds in the intestine), and metabolic, triggering biosynthesis of serine proteases and xenobiotic metabolism activation by glutathione-S-transferases and cytochrome P450 oxidoreductase. Some cuticle proteins and serine proteases were not orthologous between both species, suggesting that in A. acris, a structural resistance mechanism has been selected allowing specialization to the highly toxic host plant. Our results represent a nice example of how two phylogenetically close species diverged over recent evolutionary time related to resistance mechanisms to plant secondary metabolites.</p

DataSheet1_Insights into the differences related to the resistance mechanisms to the highly toxic fruit Hippomane mancinella (Malpighiales: Euphorbiaceae) between the larvae of the sister species Anastrepha acris and Anastrepha ludens (Diptera: Tephritidae) through comparative transcriptomics.xlsx

The Manchineel, Hippomane mancinella (“Death Apple Tree”) is one of the most toxic fruits worldwide and nevertheless is the host plant of the monophagous fruit fly species Anastrepha acris (Diptera: Tephritidae). Here we aimed at elucidating the detoxification mechanisms in larvae of A. acris reared on a diet enriched with the toxic fruit (6% lyophilizate) through comparative transcriptomics. We compared the performance of A. acris larvae with that of the sister species A. ludens, a highly polyphagous pest species that is unable to infest H. mancinella in nature. The transcriptional alterations in A. ludens were significantly greater than in A. acris. We mainly found two resistance mechanisms in both species: structural, activating cuticle protein biosynthesis (chitin-binding proteins likely reducing permeability to toxic compounds in the intestine), and metabolic, triggering biosynthesis of serine proteases and xenobiotic metabolism activation by glutathione-S-transferases and cytochrome P450 oxidoreductase. Some cuticle proteins and serine proteases were not orthologous between both species, suggesting that in A. acris, a structural resistance mechanism has been selected allowing specialization to the highly toxic host plant. Our results represent a nice example of how two phylogenetically close species diverged over recent evolutionary time related to resistance mechanisms to plant secondary metabolites.</p

Hypothetical Molecular Model of NF-YA action mode.

<p>In WT plants growing under non-stress conditions, the expression of <i>NF-YAs</i> is low due miR169-mediated post-transcriptional down-regulation, but sufficient to activate the transcription of CCAAT-box containing promoters. Upon exposure to abiotic stress, <i>NF-YA</i> levels increase due to the transcriptional activation of <i>NF-YA</i> expression (early) and to the repression of miR169 (late). NF-YAs repress early general stress response genes sequestering NF-YB/NF-YC heterodimer avoiding its interaction probably with bZIPs, and on the other hand, participating in the late down-regulation of cell wall remodeling genes. This last step could be responsible of growth arrest mediated by these TFs when plants face for long time a stressful environment.</p

Plants overexpressing <i>NF-YA</i> display reduced N starvation induced senescence.

<p>(<b>A</b>) Photograph of WT, <i>P35S:NF-YA</i> and <i>P35S:miR169nm</i> plants grown for 2 weeks on N+ (N optimum) or N− (N limited) media under standard light/dark cycle. Bar = 10 mm. (<b>B</b>) Relative chlorophyll contents of WT and overexpressing plants grown on N contrasting conditions. Total chlorophyll content was measured, normalized per gram fresh weight of sample and used to calculate the relative content (expressed as a percent of the value for the same line growing on N optimum, set to 100%). Values are means and SD of three independent experiments. Statistically significant differences using the student t-test between WT and transgenic lines are indicated (*P<0.05, **P<0.01).</p

Expression profile of mature miR169 <i>Arabidopsis</i> in seedlings exposed to different stress conditions.

<p>Seedlings were germinated in solid media for each treatment (100 mM NaCl, low N, 9.4% Sucrose or low P). Total RNA was extracted after 4, 8 and 14 days of treatment and transcript levels for miR169 determined by qRT-PCR. Because of high sequence identity quantification was made for two groups of miR169, a to g and h to n. Expression level of <i>ACT2</i> was used as internal reference. Values represents the means and error bars indicate SE of 3 independent amplification replicates.</p

<i>P35S:NF-YA</i> lines display an increased sensitivity to sucrose and ABA.

<p>(<b>A</b>) Images of twelve-day-old <i>P35S:NF-YA, P35Smir169nm</i> and WT, seedlings grown on 0.1× MS control media and on media supplemented with 9.4% sucrose or 1 µM ABA under standard light/dark conditions (16/8). A schematic representation of the distribution of the different lines on the plate is shown. (<b>B</b>) Phenotype of 14-day-old WT, <i>P35S:NF-YA</i> and <i>P35Smir169nm</i> seedlings grown in plates with medium supplemented with 9.4% sucrose and grown in vertical position in a growth chamber. Bar = 10 mm. (<b>C</b>) Relative biomass of wild-type and overexpressing plants grown on media supplemented with 9.4% sucrose (Suc) or 1 µM ABA (ABA) for twelve days. Total biomass was recorded and used to calculate the relative biomass (expressed as a percent of the value for the same line growing on control medium, set to 100%). Values are means and SD of three biological replicates statistically treated using a student <i>t</i>-test (*P<0.05, **P<0.01, ***P<0.001).</p

Expression profile of <i>Arabidopsis NF-YA</i> genes in seedlings exposed to different stress conditions.

<p>Seedlings were germinated in solid media for each treatment (100 mM NaCl, low N, 9.4% Sucrose or low P). Total RNA was extracted after 4, 8 and 14 days of treatment and transcript levels for <i>NF-YA2</i>, <i>3</i>, <i>7</i>, <i>5</i> and <i>10</i> determined by qRT-PCR. Expression level of <i>ACTIN 2 (ACT2)</i> was used as internal reference. Values represent the means and error bars indicate the standard error (SE) of 3 independent amplification replicates.</p

qRT-PCR analysis of the <i>NF-YA</i> subfamily en Pi contrasting conditions in WT and <i>hen 1-1</i> backgrounds.

<p>Transcript levels that reflect a major component of post-transcriptional regulation are in bold. +P, optimum P (1 mM) −P, low P (5 µM).</p

Z-score for overlap between early general stress response genes and those repressed in <i>PXVE:NF-YA</i> transgenic lines.

<p>The overlap of early general stress response genes reported in two previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048138#pone.0048138-Ma1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048138#pone.0048138-Walley1" target="_blank">[34]</a> and the repressed genes by NF-YAs (cutoff≤2-fold, p-value≤0.05) is given. Data obtained using the Genesect web tool available through VirtualPlant 1.2 software platform <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048138#pone.0048138-Katari1" target="_blank">[35]</a>. S, size of intersection. Significant differences (p-value<0.001) based on a Z-score overrepresented (Z-score>11) are highlighted in bold.</p