title: Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri.
abstract: Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by the bacterial phytopathogen, Xanthomonas citri subsp. citri (Xcc). GenBank houses a large collection of Expressed Sequence Tags (ESTs) enriched with transcripts generated during the defence response against this pathogen; however, there are currently no strategies in citrus to assess the function of candidate genes. This has greatly limited research as defence signalling genes are often involved in multiple pathways. In this study, we demonstrate the efficacy of RNA interference (RNAi) as a functional genomics tool to assess the function of candidate genes involved in the defence response of Citrus limon against the citrus canker pathogen. Double-stranded RNA expression vectors, encoding hairpin RNAs for citrus host genes, were delivered to lemon leaves by transient infiltration with transformed Agrobacterium. As proof of principle, we have established silencing of citrus phytoene desaturase (PDS) and callose synthase (CalS1) genes. Phenotypic and molecular analyses showed that silencing vectors were functional not only in lemon plants but also in other species of the Rutaceae family. Using silencing of CalS1, we have demonstrated that plant cell wall-associated defence is the principal initial barrier against Xanthomonas infection in citrus plants. Additionally, we present here results that suggest that H₂O₂ accumulation, which is suppressed by xanthan from Xcc during pathogenesis, contributes to inhibition of xanthan-deficient Xcc mutant growth either in wild-type or CalS1-silenced plants. With this work, we have demonstrated that high-throughput reverse genetic analysis is feasible in citrus.
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Área Virología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha, Rosario, Argentina.
authors.forename: Ramón
authors.initials: R
authors.lastname: Enrique
authors.forename: Florencia
authors.initials: F
authors.lastname: Siciliano
authors.forename: María Alejandra
authors.initials: MA
authors.lastname: Favaro
authors.forename: Nadia
authors.initials: N
authors.lastname: Gerhardt
authors.forename: Roxana
authors.initials: R
authors.lastname: Roeschlin
authors.forename: Luciano
authors.initials: L
authors.lastname: Rigano
authors.forename: Lorena
authors.initials: L
authors.lastname: Sendin
authors.forename: Atilio
authors.initials: A
authors.lastname: Castagnaro
authors.forename: Adrian
authors.initials: A
authors.lastname: Vojnov
authors.forename: María Rosa
authors.initials: MR
authors.lastname: Marano
mesh_terms: D002957:Citrus / Q000201:enzymology / Q000276:immunology* / Q000382:microbiology*; D018506:Gene Expression Regulation, Plant; D005964:Glucosyltransferases / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014974:Xanthomonas / Q000276:immunology*
chemical_list: D011135:Polysaccharides, Bacterial; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; C002563:xanthan gum