Salivary proteins of Phloeomyzus passerinii, a plant-manipulating aphid, and their impact on early gene responses of susceptible and resistant poplar genotypes

International audienceSuccessful plant colonization by parasites requires the circumvention of host defenses, and sometimes a reprogramming of host metabolism, mediated by effector molecules delivered into the host. Using transcriptomic and enzymatic approaches, we characterized salivary glands and saliva of Phloeomyzus passerinii, an aphid exhibiting an atypical feeding strategy. Plant responses to salivary extracts of P. passerinii and Myzus persicae were assessed with poplar protoplasts of a susceptible and a resistant genotype, and in a heterologous Arabidopsis system. We predict that P. passerinii secretes a highly peculiar saliva containing effectors potentially interfering with host defenses, biotic stress signaling and plant metabolism, notably phosphatidylinositol phosphate kinases which seemed specific to P. passerinii. Gene expression profiles indicated that salivary extracts of M. persicae markedly affected host defenses and biotic stress signaling, while salivary extracts of P. passerinii induced only weak responses. The effector-triggered susceptibility was characterized by downregulations of genes involved in cytokinin signaling and auxin homeostasis. This suggests that P. passerinii induces an intracellular accumulation of auxin in susceptible host genotypes, which is supported by histochemical assays in Arabidopsis. This might in turn affect biotic stress signaling and contribute to host tissue manipulation by the aphid

Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps <i>Asobara tabida</i> and <i>Leptopilina heterotoma</i>

<div><p>Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, <i>Asobara tabida</i> (Braconidae) and <i>Leptopilina heterotoma</i> (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and β-subunits also form an (αβ)₂ structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and β for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the <i>Drosophila melanogaster</i> host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps’ success.</p></div

FPLC purification profiles of AtAGA and LhAGA.

<p>A and D. FPLC profiles of <i>A</i>. <i>tabida</i> (A) and <i>L</i>. <i>heterotoma</i> (D) venom extracts at 280 nm. B and E. 12.5% SDS-PAGE analysis of each FPLC fraction for <i>A</i>. <i>tabida</i> (B) and <i>L</i>. <i>heterotoma</i> (E). Lane T, total venom extract (3 venom apparatus/well). Aspartylglucosaminidase activity measured with 20 μl of each FPLC fraction is overlay on gel pictures for AtAGA (B) and LhAGA (E). C and F. Detection of AGA on western-blots of 10 μl of each FPLC fraction of <i>A</i>. <i>tabida</i> (anti-P30 antibody, C) and <i>L</i>. <i>heterotoma</i> (anti-LhAGA, F). Only AGA positive fractions are shown.</p

Analysis of the native conformation of AtAGA and LhAGA.

<p>A. Non-reducing/reducing electrophoretic migration of <i>L</i>. <i>heterotoma</i> venom. After venom separation on a 12.5% non-reducing gel, the full lane was excised and run under reducing conditions (gel on the left). The only band that showed a migration shift (boxed) was excised, run under reducing conditions, and silver stained (Gel) or probed with the anti-LhAGA (WB) (lanes on the right). B. Glutaraldehyde cross-linking analysis of the oligomerization state of FPLC purified native AtAGA and LhAGA (% of glutaraldehyde on top of the lane). First lane: molecular weight markers.</p

Alignment of AGA sequences.

<p>Translated cDNA sequences of <i>A</i>. <i>tabida</i> (AtAGA; ACX94224) and <i>L</i>. <i>heterotoma</i> venom AGAs (LhAGA; KP888635) aligned with the sequence of human AGA (hAGA; P20933). Residues identical with hAGA are indicated by a dot. Signal peptide amino acids are in blue. Conserved residues important for structure or activity are highlighted in green, with an asterisk indicating the active-site threonine (hAGA T206). Important hAGA residues not conserved in AtAGA and/or LhAGA are highlighted in red. Cysteine residues are framed by a black box and those involved in disulfide bonds are connected with black solid lines. Potential N-glycosylation sites are in brown (hAGA N38 and N308, AtAGA N52 and N153, LhAGA N326). Residues important for hAGA phosphorylation are in purple (K177, Y178, K183 and K214).</p

Kinetic constants of AtAGA and LhAGA for AspAMC and L-asparagine.

<p>Kinetic constants of AtAGA and LhAGA for AspAMC and L-asparagine.</p

Homology modeling of AtAGA and LhAGA.

<p>A. Superposition of the active (αβ)₂ tetramer tertiary model structure of mature AtAGA (QMEAN score = 0.694; QMEAN Z-score = -0.83; colored in red) and LhAGA (QMEAN score = 0.725; QMEAN Z-score = -0.5; colored in green) with the solved structure of human AGA (hAGA; 1APY; colored in cyan). B. Spatial geometry of some of the key catalytic and binding residues of hAGA, AtAGA and LhAGA active sites.</p