Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

Characterization of a variant of Xanthomonas citri subsp. citri that triggers a host-specific defense response.

Citrus is an economically important fruit crop that is severely afflicted by Asiatic citrus bacterial canker (CBC), a disease caused by the phytopathogen Xanthomonas citri subsp. citri (X. citri). To gain insight into the molecular epidemiology of CBC, 42 Xanthomonas isolates were collected from a range of Citrus spp. across 17 different orchards in Tucumán, Argentina and subjected to molecular, biochemical, and pathogenicity tests. Analysis of genome-specific X. citri markers and DNA polymorphisms based on repetitive elements-based polymerase chain reaction showed that all 42 isolates belonged to X. citri. Interestingly, pathogenicity tests showed that one isolate, which shares >90% genetic similarity to the reference strain X. citri T, has host range specificity. This new variant of X. citri subsp. citri, named X. citri A(T), which is deficient in xanthan production, induces an atypical, noncankerous chlorotic phenotype in Citrus limon and C. paradisi and weak cankerous lesions in C. aurantifolia and C. clementina leaves. In C. limon, suppression of canker development is concomitant with an oxidative burst; xanthan is not implicated in the phenotype induced by this interaction, suggesting that other bacterial factors would be involved in triggering the defense response.

Novel demonstration of RNAi in citrus reveals importance of citrus callose synthase in defence against Xanthomonas citri subsp. citri.

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.

Relative Expression Analysis of Target Genes by Using Reverse Transcription-Quantitative PCR.

Real-time PCR is a powerful technique used for quantification of defined nucleic acid sequences. Numerous applications of this method have been described including: gene expression studies, diagnosis of pathogens, and detection of genetically modified organisms or mutations. Here, we describe a simple and efficient protocol to determine gene expression in cereals, based on real-time PCR using SYBR® Green dye. This technique provide an inexpensive alternative, since no probes are required, allowing for the quantification of a high number of genes with reduced cost.

Cereal Circular RNAs (circRNAs): An Overview of the Computational Resources for Identification and Analysis.

Circular RNAs (circRNAs) are a widespread class of endogenous noncoding RNAs and they have been studied in the past few years, implying important biological functions in all kingdoms of life. Recently, circRNAs have been identified in many plant species, including cereal crops, showing differential expression during stress response and developmental programs, which suggests their role in these process. In the following years, it is expected that insights into the functional roles of circRNAs can be used by cereal scientists and molecular breeders with the aim to develop new strategies for crop improvement. Here, we briefly outline the current knowledge about circRNAs in plants and we also outline available computational resources for their validation and analysis in cereal species.

The Genetic Transformation of Sweet Orange (Citrus sinensis L. Osbeck) for Enhanced Resistance to Citrus Canker.

Developing disease resistance is one of the most important components of any plant breeding program. Citrus traditional breeding methods (bud sport selection, crossbreeding, and other breeding channels) are a laborious task and often hampered by long juvenility, a high degree of heterozygosity, polyembryony, self-incompatibility, and abortion of reproductive organs. An interesting alternative to the classical breeding approach is the use of genetic transformation, which provides the means for adding a single agronomic trait to a plant without otherwise altering its phenotype. Agrobacterium tumefaciens-mediated transformation has been carried out with numerous hybrids and citrus species. This technique allowed us to introduce the Bs2 gene in Citrus, as well as to increase citrus canker resistance in transgenic Bs2 gene-expressing lines.

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri.

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.

Resistance to Citrus psorosis virus in transgenic sweet orange plants is triggered by coat protein-RNA silencing.

The lack of naturally occurring resistance to Citrus psorosis virus (CPsV) has demanded exploitation of a transgenic approach for the development of CPsV-resistant sweet orange plants. Transgenic sweet orange plants producing intron-hairpin RNA transcripts (ihpRNA) corresponding to viral cp, 54K or 24K genes were generated and analyzed at the molecular and phenotypic levels. Two independent CPsV challenge assays demonstrated that expression of ihpRNA derived from the cp gene (ihpCP) provided a high level of virus resistance, while those derived from 54K and 24K genes (ihp54K and ihp24K) provided partial or no resistance. The presence of small interfering RNA molecules (siRNAs) in the ihpCP transgenic sweet orange plants prior to virus challenge, indicated that CPsV resistance was due to pre-activated RNA silencing, but siRNAs accumulation level was not directly correlated to the degree of the triggered virus resistance among the different lines. However, pre-activation of the RNA-silencing machinery and a certain minimum accumulation level of siRNA molecules targeting the viral genome are key factors for creating virus-resistant plants. This is the first report of resistance in citrus plants against a negative-strand RNA virus as CPsV.

Analysis of the molecular basis of Xanthomonas axonopodis pv. citri pathogenesis in Citrus limon

Xanthomonas axonopodis pathovar citri (Xac) causes bacterial citrus canker, a serious disease of most citrus species. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease in cruciferous plants. In Xcc, cell-cell signaling is mediated by diffusible signal factor (DSF). Synthesis of DSF depends on RpfB and RpfF. DSF perception and signal transduction have been suggested to involve a two-component system comprising RpfC and RpfG. It has been proposed that these proteins participate in a signal transduction system linking changes in the environment to the synthesis of DSF and the expression of virulence genes. Although the cluster of the rpf genes in Xac has synteny with the corresponding cluster in Xcc, two genes (rpfH and rpfI) are absent in Xac. To investigate DSF-mediated regulation during Xac-Citrus limon interaction, we constructed two strains of Xac, one with a mutation in the rpfF gene, leading to an inability to produce DSF, and one with a mutation in the rpfC gene leading to an overproduction of DSF. These mutants also show decreased levels of extracellular cyclic â-(1,2)-glucans and decreased production of endoglucanase and protease extracellular enzymes. The Xac DSF-deficient rpfF and the DSF-hyper producing rpfC mutants are both severely compromised in their ability to cause canker symptoms in lemon leaves compared to the wild-type. Here we provide evidence that rpf genes in Xac are involved in controlling virulence factors mediated by DSF.