Background
Plants respond to pathogen infection through a number of gene regulatory pathways. RNA-silencing is a form of regulation where double-stranded or hairpin-structured RNA precursors give rise to small RNAs (sRNAs), which become control elements for the expression of target genes [
1‐
3]. Several types of sRNA species have been identified and characterised in plants, and their involvement in biotic stress responses have been suggested. These include microRNAs (miRNAs) [
4‐
8], phased-siRNAs (phasiRNAs) [
9‐
12], natural-antisense transcript siRNAs (nat-siRNAs) [
12‐
14], repeat-associated siRNAs (rasiRNAs) [
7] and tRNA-derived RNA fragments (tRFs) [
15,
16].
Stem pitting is a destructive symptom in fruit crops caused by various virus species.
Citrus tristeza virus (CTV) is a highly destructive, phloem limited, pathogen of citrus species, causing three different disease syndromes [
17] of which stem pitting currently is considered the highest threat to the industry [
18]. CTV belongs to the genus
Closterovirus in the family
Closteroviridae [
19]. The ~19,300 nt genome of CTV is organised into 12 open reading frames and represents the largest known plant virus genome [
20].
Due to the complex disease aetiology of CTV, which is strongly influenced by the combination of host factors and virus genotypes, the mechanism(s) behind symptom expression is poorly understood [
21,
22]. A recent study, however, has suggested the involvement of different combinations of p33, p18 and p13 expression during stem pitting symptom development [
18]. Three CTV RNA-silencing suppressors have been identified, namely p20, p23 and p25 (coat protein), all of which can play a role in symptom expression [
23].
In addition to viruses, citrus species are also affected by viroid-infections.
Citrus dwarfing viroid (CDVd), a member of the genus
Apscaviroid (family
Pospiviroidae), has been suggested for use in high-density orchards since it causes dwarfing of citrus varieties grafted onto
Poncirus trifoliata (
P. trifoliata) and its hybrids, without reducing production [
24‐
26]. The fact that citrus species are often co-infected with CTV and viroids prompted a recent study which investigated the co-infection of CTV and CDVd in their respective indicator plants, Mexican lime and etrog citron [
27]. A host-specific increase in the accumulation of CDVd was observed in the presence of CTV, along with the synthesis of CDVd-associated sRNAs. These observations were ascribed mainly to the involvement of the CTV-encoded silencing suppressor, p23. It is also interesting to note that the co-infection did not affect symptom expression under their experimental conditions [
27].
Understanding the mechanisms involved in pathogen infection and symptom expression provides the information required to study and potentially engineer disease resistance. In this study, we used a next-generation sequencing (NGS) approach to investigate the plant responses to the co-infection of CTV and CDVd in two commercial grapefruit (Citrus paradisi) cultivars, on both the sRNA and transcriptome level. Our results highlighted the association of CTV-CDVd co-infection with the expression of various genes and sRNA species, these include sRNAs involved in the regulation of plant hormones.
Discussion
In the field, citrus plants are often subjected not only to CTV infection but also to the co-infection with viroids. It is therefore necessary to understand this combined pathogen interaction with the plant host in order to improve disease resistance strategy design. In this study, healthy ‘Marsh’ and ‘Star Ruby’ grapefruit plants developed leaf cupping and stem pitting symptoms after being co-infected with CTV (genotype T3) and CDVd. These symptoms were not present in the co-infected sweet orange plants, which served as inoculation source. Although the T3 genotype is associated with increased stem pitting, previous studies have shown that CTV isolate is not the only determinant of symptom expression, but that host species also plays a role [
18,
21,
22]. The mechanism(s) that drive the severity and host-specific symptom expression remains to be elucidated.
An sRNA and transcriptome next-generation sequencing approach was followed to study the gene-regulatory pathways involved in the CTV-CDVd co-infection of grapefruit. To compensate for the limited genomic information available for grapefruit, the transcriptome data were de novo assembled to generate a case-specific grapefruit transcriptome. Since the assembled transcripts represented both coding and non-coding transcripts, they were used to identify both the precursors and targets of sRNAs.
As potential precursor source, the transcripts were first used for miRNA discovery. The miRNA registry, miRBase, currently holds no entries for grapefruit. Here we report on the identification of 60 grapefruit MIR genes along with their mature miRNAs, based on in silico prediction analysis. Many of the other sRNA reads represented homologous plant miRNAs. Once more grapefruit genome information becomes available, these homologous sequences may still prove to be true miRNAs, expressed from grapefruit MIR genes. In addition to miRNAs, phasiRNAs and PHAS transcripts were also identified, based on the assembled transcriptome, along with NATs that form the precursors of nat-siRNAs.
Many diverse “genes” were found differentially regulated in response CTV-CDVd co-infection across both grapefruit varieties. Similar to the results of previous studies on the infection of either CTV [
53‐
57] or CDVd [
58] in different citrus species and in
P. trifoliata, grapefruit metabolic, disease response, structural and phytohormone-related pathways seem to be affected by the co-infection. Genes with known involvement in plant disease responses were mostly grouped into leucine-rich repeat (which include tobacco mosaic virus resistance protein N-like) and ankyrin repeat-containing protein coding genes. The number of chloroplast-associated genes with altered expression supports the hypothesis that the CTV-CDVd co-infection influences the photosynthetic pathways of grapefruit, which was previously shown for CTV infection in sweet orange [
56,
57] and Mexican lime [
54,
59].
Many members of different endogenous sRNA species, such as miRNAs, phasiRNAs, nat-siRNAs, rasiRNAs and tRFs also showed variation in expression resulting from the co-infection. Target prediction was performed to determine the biological role of the differentially expressed sRNAs. Despite many transcripts and sRNAs showing differential expression resulting from co-infection, an inverse-regulation was only seen for three sRNA-transcript pairs. The apparent disconnect between sRNAs and their predicted targets could be due to a number of factors. First, the target prediction software used, was designed specifically for miRNA and phasiRNA target prediction and may therefore not be as effective for other sRNA species. Target prediction models remain to be developed for the other species, following the characterisation of their mechanism(s) of action. Second, although sRNA action may lead to the indirect inhibition of protein expression, the presence of any (cleaved or uncleaved) target transcript-related reads in the transcriptome dataset will count towards transcript-associated reads during differential expression analysis of the genes. Last, the relatively low read counts associated with many of the predicted target transcripts could have influenced the statistical significance of the differential expression analysis.
The combined results from this study suggested the sRNA-directed gene regulation of plant hormone pathways during CTV-CDVd co-infection in grapefruit. The expression of chloroplastic Magnesium-chelatase subunit ChlH (CHLH) showed inverse-regulation with respect to that of its regulating sRNA, across both ‘Marsh’ and ‘Star Ruby’ plants. In Arabidopsis, CHLH plays a role in chlorophyll biosynthesis, the expression of photosynthesis-related proteins, as well as abscisic acid (ABA) signal regulation [
60]. CHLH was shown to repress the expression of the disease response
WRKY40 gene in the ABA signalling pathway [
61]. Therefore, unsurprisingly,
WRKY40 showed inverse-regulation to that of CHLH resulting from the co-infection. The involvement of the ABA pathway in plant-virus response has previously been described [
62‐
64], and includes the restriction, to some extent, of virus movement through callose deposition [
62]. In addition, ABA was shown to contribute to virus-resistance through the regulation of Argonauts [
64]. The sRNA-directed regulation of CHLH during the CTV-CDVd co-infection could therefore potentially have a down-stream effect on virus resistance through ABA regulation. Cytokinin dehydrogenase 6 (CKX), on the other hand, is involved in the irreversible down-regulation of cytokinins [
65‐
67], and showed inverse-regulation with respect to that of its regulating sRNA in ‘Star Ruby’ plants. Cytokinins can stimulate plant defence response upon pathogen infection [
68], and may lead to either an enhancement in resistance [
69‐
71], or susceptibility to viral infections [
72]. The observed down-regulation of CKX may lead to an increase in cytokinin levels, contributing to the grapefruit defence response. A tissue-specific up- or down-regulation of CKX resulting from virus infection was also recently observed in Arabidopsis roots and shoots, respectively [
73].
Pathogen-derived sRNA profiles have been found to vary upon different infection. The majority of the vsiRNAs, derived from CTV, were 21 or 22 nts in length, which is common for vsiRNAs produced by many plants, including citrus [
8,
74]. The vsiRNAs also seem to favour the 3′ end of the virus. While this increase towards the 3′ end of CTV was observed before, not all genotype-host combinations showed the same pattern [
8,
74]. Factors that could influence patterns of vsiRNA synthesis include; a host-specific response, the virus genome secondary structure, virus gene expression and host-specific suppression of virus proteins.
Interestingly, when looking at the vsiRNA profile of CTV, a highly prominent hotspot was observed at the sub-genomic RNA initiation site of p23, associated with the negative strand, which indicates an area targeted by the host-response. p23 is known to play a major role in CTV-host interaction, especially as a suppressor to counter the host’s RNA-silencing response [
23]. The targeting of p23 by the host therefore adds another layer to the CTV-host interaction.
Recent studies have also investigated the plant siRNA response to viroid infection [
75‐
79]. While the majority of CDVd-derived vd-siRNAs were 22, 21 or 24 nts in length respectively, the observed size-distribution may be tissue dependent [
76,
77]. As was seen for CTV, an sRNA hotspot was observed associated with an area on the negative-strand of CDVd. A similar hotspot was previously observed for another member of the same genus,
Potato spindle tuber viroid, in tomato [
75,
78]. The implication of the sRNA targeting of this specific area of the viroid remains to be elucidated.