Comparison of Current Methods for Signal Peptide Prediction in Phytoplasmas.

Although phytoplasma studies are still hampered by the lack of axenic cultivation methods, the availability of genome sequences allowed dramatic advances in the characterization of the virulence mechanisms deployed by phytoplasmas, and highlighted the detection of signal peptides as a crucial step to identify effectors secreted by phytoplasmas. However, various signal peptide prediction methods have been used to mine phytoplasma genomes, and no general evaluation of these methods is available so far for phytoplasma sequences. In this work, we compared the prediction performance of SignalP versions 3.0, 4.0, 4.1, 5.0 and Phobius on several sequence datasets originating from all deposited phytoplasma sequences. SignalP 4.1 with specific parameters showed the most exhaustive and consistent prediction ability. However, the configuration of SignalP 4.1 for increased sensitivity induced a much higher rate of false positives on transmembrane domains located at N-terminus. Moreover, sensitive signal peptide predictions could similarly be achieved by the transmembrane domain prediction ability of TMHMM and Phobius, due to the relatedness between signal peptides and transmembrane regions. Beyond the results presented herein, the datasets assembled in this study form a valuable benchmark to compare and evaluate signal peptide predictors in a field where experimental evidence of secretion is scarce. Additionally, this study illustrates the utility of comparative genomics to strengthen confidence in bioinformatic predictions.

Flavescence Dorée Phytoplasma Has Multiple ftsH Genes that Are Differentially Expressed in Plants and Insects.

Flavescence dorée (FD) is a severe epidemic disease of grapevines caused by FD phytoplasma (FDP) transmitted by the leafhopper vector Scaphoideus titanus. The recent sequencing of the 647-kbp FDP genome highlighted an unusual number of genes encoding ATP-dependent zinc proteases FtsH, which have been linked to variations in the virulence of "Candidatus Phytoplasma mali" strains. The aims of the present study were to predict the FtsH repertoire of FDP, to predict the functional domains and topologies of the encoded proteins in the phytoplasma membrane and to measure the expression profiles in different hosts. Eight complete ftsH genes have been identified in the FDP genome. In addition to ftsH6, which appeared to be the original bacterial ortholog, the other seven gene copies were clustered on a common distinct phylogenetic branch, suggesting intra-genome duplication of ftsH. The expression of these proteins, quantified in plants and insect vectors in natural and experimental pathosystems, appeared to be modulated in a host-dependent manner. Two of the eight FtsH C-tails were predicted by Phobius software to be extracellular and, therefore, in direct contact with the host cellular content. As phytoplasmas cannot synthesize amino acids, our data raised questions regarding the involvement of FtsH in the adaptation to hosts via potentially enhanced recycling of phytoplasma cellular proteins and host protein degradation.

Export of salicylic acid from the chloroplast requires the multidrug and toxin extrusion-like transporter EDS5.

Salicylic acid (SA) is central for the defense of plants to pathogens and abiotic stress. SA is synthesized in chloroplasts from chorismic acid by an isochorismate synthase (ICS1); SA biosynthesis is negatively regulated by autoinhibitory feedback at ICS1. Genetic studies indicated that the multidrug and toxin extrusion transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) of Arabidopsis (Arabidopsis thaliana) is necessary for SA accumulation after biotic and abiotic stress, but so far it is not understood how EDS5 controls the biosynthesis of SA. Here, we show that EDS5 colocalizes with a marker of the chloroplast envelope and that EDS5 functions as a multidrug and toxin extrusion-like transporter in the export of SA from the chloroplast to the cytoplasm in Arabidopsis, where it controls the innate immune response. The location at the chloroplast envelope supports a model of the effect of EDS5 on SA biosynthesis: in the eds5 mutant, stress-induced SA is trapped in the chloroplast and inhibits its own accumulation by autoinhibitory feedback.

Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis.

Salicylic acid (SA) is an important mediator of plant defense response. In Arabidopsis (Arabidopsis thaliana), this compound was proposed to derive mainly from isochorismate, itself produced from chorismate through the activity of ISOCHORISMATE SYNTHASE1 (ICS1). Null ics1 mutants still accumulate some SA, suggesting the existence of an enzymatic activity redundant with ICS1 or of an alternative ICS-independent SA biosynthetic route. Here, we studied the role of ICS2, a second ICS gene of the Arabidopsis genome, in the production of SA. We have shown that ICS2 encodes a functional ICS enzyme and that, similar to ICS1, ICS2 is targeted to the plastids. Comparison of SA accumulation in the ics1, ics2, and ics1 ics2 mutants indicates that ICS2 participates in the synthesis of SA, but in limited amounts that become clearly detectable only when ICS1 is lacking. This unequal redundancy relationship was also observed for phylloquinone, another isochorismate-derived end product. Furthermore, detection of SA in the double ics1 ics2 double mutant that is completely devoid of phylloquinone provides genetic evidence of the existence of an ICS-independent SA biosynthetic pathway in Arabidopsis.

FiRe and microarrays: a fast answer to burning questions.

FiRe is a user-friendly Excel macro designed to survey microarray data rapidly. This software interactively assembles data from different experiments and produces lists of candidate genes according to patterns of gene expression. Furthermore, macros bundled with FiRe can compare lists of genes, merge information from different spreadsheets, link candidates to information available from web-based databases, and produce heat-maps for easy visualization of microarray data. FiRe is freely available at http://www.unifr.ch/plantbio/FiRe/main.html .

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1.

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.