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

Abstract

SummaryCitrus 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 H 2 O 2 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.

The Coiled-Coil and Nucleotide Binding Domains of BROWN PLANTHOPPER RESISTANCE14 Function in Signaling and Resistance against Planthopper in Rice

“…Several RLCKs are thought to play pivotal roles in plant defense (Martin et al, 1994;Swiderski and Innes, 2001;Zhang et al, 2010;Shi et al, 2013;Liu et al, 2011;Feng et al, 2012;Shinya et al, 2014;Yamaguchi et al, 2013;Dubouzet et al, 2011;Wang et al, 2015b;Zhou et al, 2016) and are thought to be involved in connecting signal perception by RLKs in the plasma membrane to downstream intracellular signal transduction systems. Callose synthases such as LOC_Os01g67364.1 perform callose biosynthesis in plants (Xie et al, 2011), and there is considerable evidence that they function in preventing penetration by herbivores (Enrique et al, 2011;Blümke et al, 2013Blümke et al, , 2014Ellinger et al, 2013). We found that the promoters of RLCK281 and LOC_Os01g67364.1 were associated with WRKY46 and WRKY72; the presence of the CC, NB, and CN domains or FL BPH14 enhanced the association between the WRKYs and the promoters of RLCK281 and LOC_Os01g67364.1 in rice.…”

Induced resistance in sweet orange against Xanthomonas citri subsp. citri by hexanoic acid

“…Lee et al (2009) proposed that callose also contributes to resistance against invading bacterial pathogens by providing a physical barrier. Recently, Enrique et al (2011) demonstrated in callose-silenced citrus plants, that reducing callose levels weakened this barrier and led to enhanced Xcc susceptibility compared to wild-type plants. Moreover, Yun et al (2006) found that callose is required for resistance to Xcc and that suppressing callose deposition induces susceptibility to Xcc in N. benthamiana and Arabidopsis.…”

“…Reverse transcription and real-time quantitative PCR (RT-qPCR) were performed as previously described (Francis et al, 2009). The primers used in the RT-qPCR were pth and PR2 previously described by Francis et al (2009), Callose synthase 1 (CALs1) described by Enrique et al (2011) and Allene oxide synthase (AOS) and PR5 described by Fern andez-Crespo et al (2012). Actin and 18S gene expression were used as an internal standard (Yan et al, 2012) (Supplementary table 1: primer sequences).…”

“…Once the pathogen attack is recognized by the plant, it activates cell wall-associated defense such as the callose deposition and the rapid accumulation of reactive oxygen species (ROS) which is the principal initial barrier against Xanthomonas infection in citrus plants (Enrique et al, 2011). In addition to these physical barriers, innate plant defenses trigger a broad spectrum of metabolic and hormonal responses (Durrant and Dong, 2004;Lorenzo et al, 2004).…”

Hexanoic acid provides long-lasting protection in ‘Fortune’ mandarin against Alternaria alternata

“…We used the RT-qPCR conditions that were previously described by Flors et al [16]. The primers that were used in the RT-qPCR were CALs1 as described by Enrique et al [17], PGIP as described by Llorens et al [15] and AOS and GAPDH (as an internal standard) as described by Fernandez-Crespo et al [18].…”