Editorial: RNAi Based Pesticides

3openInternationalInternational coauthor/editoropenAndrás Székács; Azeddine Si Ammour; Michael L. MendelsohnSzékács, A.; SI AMMOUR, A.; Mendelsohn, M.L

Signs of Silence: Small RNAs and Antifungal Responses in Arabidopsis thaliana and Zea mays

Plant small RNAs (sRNAs) are pivotal regulators of gene expression, which are crucial in maintaining genome integrity and flexibility during development, abiotic and biotic stress responses. Current evidence suggests that sRNAs might be inherent to the sophisticated plant innate immune system battling bacteria. However, the role of sRNAs during antifungal plant defences is less clear. Therefore, this chapter investigates the sRNA‐mediated plant antifungal responses against the hemibiotrophic fungi Colletotrichum higginsianum and Colletotrichum graminicola in their respective compatible hosts Arabidopsis thaliana and Zea mays. A phenotypic and metabolomic analysis of A. thaliana sRNA mutants in response to C. higginsianum infection was performed, showing a hormonal and metabolic imbalance during fungal infection in these plants. To find whether fungal-induced sRNA could directly regulate defence genes in an agricultural important plant model, the expression of maize miRNAs in response to C. graminicola leaf and root infections was investigated. The results revealed the tissue‐specific local and systemic adaptation of the miRNA transcriptome, where only a few miRNAs were targeting defence pathways. The general picture presented here points towards a role of sRNAs as fine‐tuners of genetic and metabolomic defence response layers. This chapter also further discusses the potential of utilizing sRNA‐based fungal control strategies

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interaction

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections

Quantification of induced resistance against <i>Phytophthora</i> species expressing GFP as a vital marker: β-aminobutyric acid but not BTH protects potato and Arabidopsis from infection

Induced resistance was studied in the model pathosystem Arabidopsis-Phytophthora brassicae (formerly P. porri) in comparison with the agronomically important late blight disease of potato caused by Phytophthora infestans. For the quantification of disease progress, both Phytophthora species were transformed with the vector p34GFN carrying the selectable marker gene neomycine phosphotransferase (nptII) and the reporter gene green fluorescent protein (gfp). Eighty five per cent of the transformants of P. brassicae and P. infestans constitutively expressed GFP at high levels at all developmental stages both in vitro and in planta. Transformants with high GFP expression and normal in vitro growth and virulence were selected to quantify pathogen growth by measuring the in planta emitted GFP fluorescence. This non-destructive monitoring of the infection process was applied to analyse the efficacy of two chemical inducers of disease resistance, a functional SA-analogue, benzothiadiazole (BTH), and β-aminobutyric acid (BABA) which is involved in priming mechanisms of unknown nature. BABA pre-treatment (300 µm) via soil drench applied 24 h before inoculation completely protected the susceptible Arabidopsis accession Landsberg erecta (Ler) from infection with P. brassicae. A similar treatment with BTH (330 µm) did not induce resistance. Spraying the susceptible potato cultivar Bintje with BABA (1 mm) 2 days before inoculation resulted in a phenocopy of the incompatible interaction shown by the resistant potato cultivar Matilda while BTH (1.5 mm) did not protect Bintje from severe infection. Thus, in both pathosystems, the mechanisms of induced resistance appeared to be similar, suggesting that the Arabidopsis-P. brassicae pathosystem is a promising model for the molecular analysis of induced resistance mechanisms of potato against the late blight disease

Molecular analysis of the arabidopsis-Phytophthora pathosystem

Afin de mieux comprendre l´interaction Phytophthora-plante, nous avons développé un nouveau pathosystème: Arabidopsis thaliana-Phytophthora porri. Jusqu´à présent, Phytophthora infestans, qui a causé famine et désolation en Irlande voilà 150 ans, a été le mieux étudié. Etudier le pathosystème Pomme de terre-Phytophthora infestans a, sans aucun doute, des avantages surtout parce que la pomme de terre est très cultivée de par le monde. Toutefois, utiliser Arabidopsis comme plante modèle pour des études à l´échelle moléculaire des réactions incompatibles et compatibles, nous permettra d'élaborer des stratégies pour mieux lutter contre Phytophthora. Si différentes lignées ou mutants d´Arabidopsis sont infectés avec P. porri, différents phénotypes peuvent être obtenus. En bref, Columbia est résistant, Landsberg erecta est susceptible et le mutant pad2-1 est hypersusceptible. Ce mutant n´accumule ni l´acide salicylique ni la camalexine. En outre, une étude menée avec différents mutants déficients en production d´acide salicylique, d´acide jasmonique ou de camalexine a montré qu´aucun de ces composé chimiques n´est important pour Arabidopsis pour résister à une infection contre Phytophthora. Toutefois, la fonction de la protéine PAD2 est absolument requise (Chapitre II). Il était alors, nécessaire de vérifier si la résistance est réellement indépendante de l´acide salicylique. Pour cela, des inductions par voie chimique ont été réalisées avec l´acide salicylique et son analogue le BTH. L´acide -aminobutyrique (BABA) a été aussi utilisé dans cette étude car il a été démontré qu´il induit la résistance chez la tomate et la pomme de terre contre Phytophthora. Pour estimer le degré de protection in planta d´une manière non destructive, nous avons transformé P. porri et P. infestans de manière à ce qu´ils expriment une protéine vert fluorescent, la GFP. Cela permet une estimation du degré d´infection assez facile. De cette manière, nous avons estimé la protection que BABA confère à Arabidopsis et la pomme de terre contre Phytophthora à respectivement, 100% et 97%. En revanche, le BTH n´induit pas de résistance significative confirmant ainsi nos conclusions antérieures (Chapitre III). Il est clair que cette nouvelle voie de signalisation est contrôlée par PAD2 et que cette voie est indépendante de l´acide salicylique. Nous avons utilisé des "oligonucleotides-based arrays" pour trouver des gènes pouvant servir de marqueurs à cette voie. Cette analyse s´est limitée aux gènes transcrits de importante dans la réaction incompatible mais pas chez le mutant pad2-1. En utilisant, le logiciel GeneCluster 1.0, nous avons identifié sept gènes présentant le profil recherché. Quatre d´entre eux ont une fonction inconnue: une putative protéine, une putative "disease resistance" protéine, une putative tyrosine aminotransferase et une protéine inconnue. Les trois autres gènes ont été déjà décrits dans la littérature: une serine/threonine kinase, le géne AIG1 (avirulence induced gene) et le gène PAD4. Une analyse plus poussée a montré que l´induction de ces sept gènes par P. porri est dépendante de l´acide salicylique. Donc aucun de ces gènes ne peut être utilisé comme marqueur pour la nouvelle voie de signalisation contrôlée par PAD2 (Chapitre IV). Finalement, pour effectuer des études génétiques avec P. porri nous avons déterminé la taille de son génome par les techniques "flow-cytometry" et reconstruction génomique. Nous avons estimé la taille du génome de P. porri à 110 10 Mbp ce qui représente à peu prés la moitié de la taille du génome de P. infestans (Chapitre V).In order to better understand the Phytophthora-plant interaction, we have developed a new pathosystem: Arabidopsis thaliana-Phytophthora porri. At present, the best studied Phytophthora species is P. infestans which caused the dramatic Irish late blight epidemics 150 years ago. Studying the pathosystem Phytophthora infestans-Solanum tuberosum has certain advantages mainly because potato is an important crop plant. However, using Arabidopsis as a plant model for molecular studies of both the incompatible interaction and the compatible interaction, will help us to find new strategies to control Phytophthora. When different accessions or mutants of Arabidopsis are infected with P. porri, different phenotypes can be obtained. For instance, the accession Columbia is resistant, the accession Lansdberg erecta is susceptible and the mutant pad2-1 is hypersusceptible. The mutant pad2-1 is deficient in both camalexin and salicylic acid accumulation. Moreover, using different mutants impaired in SA, jasmonate, ethylene or camalexin production showed that neither SA nor jasmonate/ethylene nor camalexin are necessary for resistance towards Phytophthora but the function of PAD2 is absolutely required (Chapter II). It was therefore necessary to check if the resistance is completely SA-independent. Induced resistance using the SA analog BTH was performed. ß-aminobutyric (BABA) acid was also used in this study because it was previously shown that this compound can induce resistance toward P. infestans in tomato and potato. To estimate the degree of protection in planta in a non-destructive manner, we have transformed both P. porri and P. infestans with the visible marker green fluorescent protein (GFP). This allows an easy scoring of the infection process by measuring the fluorescence emitted by transgenic Phytophthora in planta. In this way, we have estimated a protection of 100% in the BABA treated Arabidopsis plants and 97% in the BABA treated potato. In contrast, BTH did not induce significant resistance, confirming our previous conclusions (Chapter III). It is clear that establishment of the resistance is controlled by PAD2 in a SAindependent. We aimed by using oligonucleotide-based arrays to find marker genes for this new pathway. By profiling the transcriptome of both the resistant accession Columbia and the hypersusceptible mutant pad2-1 we aimed to find those markers. We restricted the analysis to genes which are upregulated in the incompatible interaction with accession Columbia but not in pad2-1. Using the GeneCluster 1.0 software seven genes were identified. Four have an unknown function: a putative protein, a putative disease resistance protein, a putative tyrosine aminotransferase and an unknown protein. The three others were already described: a serine/threonine kinase-like protein, an avirulence induced gene AIG1 and the gene PAD4. A closer analysis revealed that the induction of all the seven genes by inoculation with Phytophthora porri is SA-dependent. Therefore, none of them is a marker gene for the new PAD2-controlled signaling pathway (Chapter IV). Finally, as a basis for its genetic analysis the genome size of P. porri was determined by flow cytometry and genomic reconstruction analysis. We have determined the genome size of P. porri to be 110 10 Mbp which is about half of the genome size of P. infestans (Chapter V)