Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

Plants can perceive a wide range of biotic attackers and respond with targeted induced defenses. Specificity in plant non-selfrecognition occurs either directly by perception of pest-derived elicitors or indirectly through resistance protein recognition of host targets that are inappropriately proteolyzed. Indirect plant perception can occur during interactions with pathogens, yet evidence for analogous events mediating the detection of insect herbivores remains elusive. Here we report indirect perception of herbivory in cowpea (Vigna unguiculata) plants attacked by fall armyworm (Spodoptera frugiperda) larvae. We isolated and identified a disulfide-bridged peptide ( ؉ ICDINGVCVDA ؊ ), termed inceptin, from S. frugiperda larval oral secretions that promotes cowpea ethylene production at 1 fmol leaf ؊1 and triggers increases in the defenserelated phytohormones salicylic acid and jasmonic acid. Inceptins are proteolytic fragments of chloroplastic ATP synthase ␥-subunit regulatory regions that mediate plant perception of herbivory through the induction of volatile, phenylpropanoid, and protease inhibitor defenses. Only S. frugiperda larvae that previously ingested chloroplastic ATP synthase ␥-subunit proteins and produced inceptins significantly induced cowpea defenses after herbivory. Digestive fragments of an ancient and essential plant enzyme, inceptin functions as a potent indirect signal initiating specific plant responses to insect attack.elicitor ͉ guard hypothesis ͉ indirect perception ͉ insect herbivory ͉ plant defense A mechanistic understanding and targeted improvement of plant resistance traits are recognized as essential in combating yield losses from crop pests. Plants can perceive and defensively respond to attack either directly by impeding pest growth or indirectly by promoting advantageous interactions with beneficial organisms (1-7). Great progress has been made in the identification of plant receptor-like kinase families mediating perception of biotic attack and the subsequent activation of signal transduction cascades spanning interactions of GTP binding proteins, mitogen-activated protein kinases, phytohormones, transcription factors, and ultimately induced biochemical defenses (2,8). Despite these advances, relatively few candidate elicitors and ligands responsible for the initiation and specificity of induced plant defenses to pest attack have been identified (1, 2). This void is especially acute in the case of insect herbivore perception and is surprising given both the significance of plant-insect interactions in arthropod and angiosperm evolution and the role of insects in facilitating plant pathogen entry (9, 10).Induced plant defenses are initiated in part by the direct perception of elicitors derived from offending organisms. For example, maize (Zea mays) and tobacco (Nicotiana attenuata) perceive insect attack through the direct detection of fatty acid amino acid conjugate (FAC) elicitors present in insect oral secretions (OS). Plants respond with indirect defenses in the form of indu...

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“…All reactions were done in triplicate. Molecule numbers per microgram of total RNA were calculated by using a standard curve technique (37).…”

Section: Methodsmentioning

confidence: 99%

Fragments of ATP synthase mediate plant perception of insect attack

Schmelz

Carroll

LeClere

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

359

265

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“…RT-qPCR was not used to validate gene expression due to the prerequisite for highly characterized sequence data (Unamba et al, 2015). A number of criteria need to be met for qPCR reference genes to be regarded as reliable (Chervoneva et al, 2010), importantly, traditional "housekeeping" genes (such as b-actin and 18S) have been extensively acknowledged as unstable in numerous biological systems (Gorzelniak et al, 2001;Solanas et al, 2001;Glare et al, 2002;Raaijmakers et al, 2002;Brunner et al, 2004;Gonçalves et al, 2005;Nicot et al, 2005;González-Verdejo et al, 2008;Barsalobres-Cavallari et al, 2009;Paolacci et al, 2009;Xu et al, 2012b;Jiang et al, 2014;Llanos et al, 2015). While many of these cited studies establish expression stability of more appropriate nontraditional reference genes, the scale and complexity of multiple nonmodel plant cultivars (in this extralaboratory research) makes such establishment problematic without using RNA-Seq itself to establish transcriptome-wide expression.…”

Section: De Novo Assemblymentioning

confidence: 99%

Comparative Transcriptomic Approaches Exploring Contamination Stress Tolerance in Salix sp. Reveal the Importance for a Metaorganismal de Novo Assembly Approach for Nonmodel Plants

et al. 2016

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Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, a/b-tubulin, and elongation factor 1-a. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system.Coppiced willows have the ability to produce high biomass yields in temperate regions under challenging conditions and have positive impacts on biodiversity (Labrecque et al

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“…The expressions of these genes are expected to undergo little variation under diverse conditions. Actin, tubulin, ubiquitin, and glyceraldehyde-3-phosphate dehydrogenase are widely used reference genes in many plant species (Gonçalves et al 2005;Jain et al 2006;Nicot et al 2005;Wan et al 2010). However, the expression levels of these genes can be changed across different samples or by external factors.…”

Section: Introductionmentioning

confidence: 99%