CTV mild strain B2 (T30 genotype, Florida) and severe strain B6 (mixed genotype, SY568, California; Vives et al., 2005; Ruiz-Ruiz et al., 2006) are maintained in planta as part of the Exotic Pathogens of Citrus Collection (EPCC) at the USDA-ARS Beltsville Agricultural Research Center (BARC) in Beltsville, MD. Both CTV-B2 and CTV-B6 were graft-inoculated into ten “Valencia” sweet orange seedlings simultaneously. Ten trees were mock-inoculated with their own buds as the healthy control.

Inoculated plants were tested for the presence of CTV specific amplicons by RT-PCR as described (Roy et al., 2010). RNA extracts were prepared for RNA-Seq immediately after the trees became RT-PCR positive and before any symptoms developed from three young, soft, not fully expanded leaves of uniform size. Leaves were frozen in liquid nitrogen and kept at −80°C until the extraction of RNA and assessment for quantity and quality with Qubit (Thermo-Fisher, Pittsburgh, PA) and Bioanalyzer (Agilent, Santa Clara, CA) instruments. Three qualified RNA replicates, each of three leaves from different plants, for CTV-B2/CTV-B6 and healthy citrus were sent to Otogenetics (Norcross, GA, USA) for paired-end sequencing with the Illumina HiSeq 2500 platform.

Raw reads obtained by Illumina HiSeq 2500 were filtered to exclude low complexity reads. Clean reads from the nine libraries were aligned to the reference genome (Xu et al., 2013) with Bowtie (Langmead, 2010) and fold changes (log₂ FC) for each transcript were evaluated by DEseq2 (Love et al., 2014). Differentially expressed transcripts (DETs) were filtered with cut-off values, Padj ≤ 0.1, |log₂ FC| ≥ 1 and e-value ≤ e⁻⁵. DETs involved in pathways were enriched with Panther (Mi et al., 2009) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) data bases. A summary of gene expression patterns was visualized with MapMan software (Thimm et al., 2004). Twenty genes were selected and transcripts were amplified by RT-qPCR to validate the RNASeq analysis as described (Fu et al., 2016). Primers used for RT-qPCR were designed by Real-time PCR online tool (Integrated DNA Technology, IDT, Table S1).

In order to estimate the relative concentration of CTV-B2 and CTV-B6 genomes in co-infected plants, RNA-Seq libraries from trees co-inoculated with CTV-B2 and CTV-B6 and mock inoculated trees were searched for sequences homologous to the p23 protein of reference CTV T30 genome (AF260651) and the reference CTV T318A genome (DQ151548). The 100 bp paired end reads were mapped to the reference genomes under stringent conditions, allowing a maximum of 1 mismatch per read duo to the high homology between the p23 protein of CTV-B2 and CTV-B6.

Ten doubly-inoculated trees became positive for both mild CTV-B2 and severe CTV-B6 based on RT-PCR after 2–4 months (Figure 1). Symptoms developed 5–6 months after inoculation. Trees infected with CTV-B2/CTV-B6 were much smaller and stunted than the healthy controls, with leaf chlorosis, vein corking, and vein curling (Figure 2) increasing as time passed. Symptoms of the co-inoculated plants 12 months after co-inoculation were indistinguishable from those produced by plants infected with CTV-B6 alone. In the plants co-inoculated with CTV-B2 and CTV-B6, the p23 transcript of CTV-B6 was found 300 times more frequently than the CTV-B2 homolog. Sequences homologous to p23 were not found in the mock-inoculated controls, nor when other libraries that contained only CTV-B2 or CTV-B6 were searched for the heterologous p23 sequence.

38 to 47 million raw reads were obtained from each inoculated tree with approximate 61% (average of three replicates) of these reads successfully aligned to the C. sinensis reference genome (Xu et al., 2013; Figure S1). Compared with mock-inoculated healthy trees, a total of 767 transcripts were differentially expressed: 411 were induced and 356 were repressed (Padj ≤ 0.1; Figure S2). DETs were found to be enriched for different biological processes (level 2) with Panther based on orthologs of Arabidopsis thaliana. The distribution of functional categories for up- and down-regulated genes was similar: metabolic processes, cellular processes, localization, biological regulation, and response to stimulus (Figure 3). The set of down-regulated genes had relatively more genes related to cellular component organization/biogenesis but less for localization (Figure 3). In an overview of metabolic and biotic stress pathways, genes related to light reactions, oxidative pentose phosphate (OPP), tricarboxylic acid cycle (TCA), tetrapyrrole synthesis, nucleotide metabolism, carbohydrate, and lipid metabolism were mostly repressed (Figure 4), whereas the genes associated with cell wall synthesis, secondary and hormone metabolism, pathogenesis-related (PR) proteins, and signaling were mostly induced (Figure 5). A full list of differentially expressed transcripts is shown in more detail (Table S2).

The expression levels of selected DETs ranged from −2 to 8 (log₂ FC). Similar expression patterns were obtained for all evaluated transcripts by both RNA-seq and RT-qPCR techniques. The dissociation pattern and single peak obtained after RT-qPCR verified the specificity of RT-qPCR primers (Figure S3). A high Spearman's rho value (0.82) indicated good correlation of gene expression between the RNA-seq and RT-qPCR results and confirmed the reliability and accuracy of the RNA-seq data in our study (Figure 6).

The expression of several photosynthesis-related genes was down-regulated in response to CTV-B2/CTV-B6. These genes included LHCA4 (light-harvesting chlorophyll-protein complex I subunit A4), PPL1 (PsbP-like protein 1), thylakoid lumenal 20 kDa protein, PSAD-1 (photosystem I subunit D-1), photosystem II reaction center PsbP family protein, and PTAC14 (plastid transcriptionally active14; Table 1). Genes in the tetrapyrrole synthesis pathway were also repressed, including GSA2 (glutamate-1-semialdehyde 2, 1-aminomutase 2), HEMC (hydroxymethylbilane synthase), HEME2 (uroporphyrinogen decarboxylase), and HEMF1 (coproporphyrinogen oxidase; Table 1).

Genes encoding enzymes for nucleotide metabolism PYR4 (pyrimidine 4), NDPK2 (nucleoside diphosphate kinase 2), and RNR1 (ribonucleotide reductase 1) were repressed. Genes related to DNA synthesis were also down-regulated, as were some genes that encode DNA repair proteins MCM2 (DNA replication licensing factor), MCM3, FAS1 (fasciata 1), RPA70B (rpa70-kda subunit B), PRL (prolifera), and genes for several histones, including H3, H4, and HTA7 (Table 1).

A large group of genes encoding ribosomal proteins were repressed, including components of the 30S, 40S, 50S, and 60S subunits, indicating an extensive reprogramming of ribosomal synthesis (Table 2). Ribosomal proteins (RPs) play roles in metabolism, growth and cell division, but also function in developmental processes as regulatory components (Byrne, 2009).

The expression of a majority of genes for cell wall metabolism was up-regulated: Genes encoding CESA9 (cellulose synthase A9) and PRP4 (cell wall proline-rich protein 4), as well as XTH28 (xyloglucan endotransglycosylase) and XTH30 were all up-regulated and the expression level of EXLB3 (expansin-like B3 precursor) was up-regulated ~7 fold (Table 3).

Transcription of genes related to transport and metal binding were largely up-regulated. These included transporters for nucleotides, sugars, amino acids, sulfate, phosphate, peptides, and oligopeptides and ABC transporters, such as, AAP6 and AAP7 (amino acid trans membrane transporter), AST56 and AST68 (sulfate transporter), NRT1.1 (nitrate transporter), ZIP1, ZIP5, and ZIP11 (zinc ion trans membrane transporter; Table 3). Transcripts encoding phloem protein PP2-B13 were somewhat down-regulated in response to co-infection with CTV-B2/CTV-B6, but were strongly down-regulated by CTV-B2 alone, whereas transcripts for PP2-B15 were slightly up-regulated by single infection with CTV-B6 (Fu et al., 2016).

Genes involved in the synthesis of secondary metabolites and hormone metabolism were induced, such as, TPS21 (terpene synthase 21), CER3 (ceriferum 3), TT4 (transparent testa 4), AFB5 (auxin F-Box protein 5), GA5 (gibberellin 20-oxidase), EIN4 (ethylene insensitive 4), and ERS1 (ethylene response sensor 1). Genes for biotic stress associated proteins RLP21 (Receptor Like Protein 21), CHIB1 (acidic endochitinase) and EDS1 (enhanced disease susceptibility 1), and disease resistance proteins in the CC-NBS-LRR class were also induced (Table 3). Genes for receptor kinases, calcium and light related signaling were mostly induced, such as, RLP1 (receptor like protein 1), RLP6, and RLP15, CSE101 (callus expression of rbcs 101), IQD23 (IQ-domain 23) binding calmodulin, and FRS5 (FAR1-related sequence 5) binding zinc ions (Table 3).

Both strains of CTV were able to establish infections in the inoculated sweet orange plants, indicating that simultaneous inoculation of CTV-B2 and CTV-B6 did not prevent infection and establishment of either strain in sweet orange. We isolated RNA from the inoculated plants after infection was established but before symptoms were expressed. The assessment of gene expression prior to the onset of visible symptoms allowed identification of changes in gene expression that led to the symptoms that were subsequently observed. Symptoms of infection by strain CTV-B2 are very mild and difficult to observe. However, in our doubly inoculated plants the symptoms were extensive and severe. CTV-B6 has a complex genome based on components of several genotypes (Ruiz-Ruiz et al., 2006), and the nucleotide sequence of the p23 gene is 98.5% identical to the reference strains T318A. In our co-inoculated plant, the p23 transcript of CTV-B6 was found much more frequently than the CTV-B2 homolog, consistent with the symptoms induced in the plants (Ghorbel et al., 2001). In cases where strains are simultaneously present in sweet orange, the genotype of the severe component often becomes dominant (Sambade et al., 2007). This is evidence that CTV-B6 is the dominant strain when trees are co-infected with mild CTV-B2 and the severe, virulent CTV-B6, and that CTV-B2 is not able to protect sweet orange from CTV-B6.

Genes that encode proteins that are components of both the light harvesting complex and that contribute to the assembly of tetrapyrroles and chlorophyll were down regulated. In addition to photosynthesis, tetrapyrroles play crucial roles in a broad range of biological processes including respiration and assimilation of nitrogen/sulfur in higher plants (Tanaka et al., 2011). In our study, genes associated with photosynthesis and tetrapyrrole metabolism were mostly down-regulated in response to CTV-B2/CTV-B6. HEMC, HEME2, and HEMF1 are enzyme intermediates in tetrapyrrole synthesis and their down-regulation indicates a disturbance of tetrapyrrole synthesis, as well as chlorophyll synthesis in host plants, because chlorophyll biosynthesis has been suggested to depend upon a balance between the methyl erythritol phosphate and tetrapyrrole pathways (Kim et al., 2013). The PsbP protein is indispensible for the regulation/stabilization (Ifuku et al., 2005) and photo autotrophy (Yi et al., 2007) of the PSII complex, as well as normal thylakoid membrane architecture (Yi et al., 2009) in higher plants. PsbP-like proteins (PPLs) are PsbP homologs and PPL1 is required for efficient repair of photo damaged PSII in higher plants (Ishihara et al., 2007). These alterations in gene expression patterns are consistent with altered thylakoid membrane structure and function in response to CTV infection. The decrease of several photosynthetic proteins (i.e., PSAD, PsbP, and LHCA) would also cause defects in photosynthetic performance (Romani et al., 2012). Symptoms such as, yellowing and chlorosis of leaves caused by CTV-B6 or CTV-B2/CTV-B6 may be due in part to the decrease of the PsbP andPPLs, as observed in experiments with PsbP-deficient tobacco (Nicotiana tabacum) which had pale-green-colored leaves (Yi et al., 2009). The decrease in the levels of these proteins limits plant growth as there is less carbohydrate synthesized as a result of the repressed photosynthesis.

Iron metabolism was also dramatically altered in response to infection by CTV-B2 and CTV-B6, which likely contributed to the alterations photosynthetic processes observed. As we observed with infection of citrus with CTV-B2/CTV-B6, genes related to photosynthesis and tetrapyrrole metabolism were also dramatically repressed in Arabidopsis leaves in response to iron (Fe) deficiency (Rodríguez-Celma et al., 2013). Iron has crucial effects on respiration and tetrapyrrole metabolism and its deficiency compromises chlorophyll synthesis, causing chlorosis in developing leaves and decreased photosynthetic activity. The iron deficiency may be exacerbated by the decline of feeder roots induced by CTV-B6, which limit the uptake of iron from the rhizosphere.

The overwhelming down-regulation of genes related to DNA synthesis is also in concert with iron deficiency as iron is required in many enzymatic reactions in the DNA replication pathway. Hexameric mini chromosome maintenance proteins (MCMs) have important roles in DNA replication in plants (Ni et al., 2009; Herridge et al., 2014) and some of them are critical for DNA unwinding. MCM2 modulates root meristem function in Arabidopsis (Ni et al., 2009). The down regulation of MCM2 is consistent with the decline in feeder roots seen as the CTV syndrome develops. MCM2 was also down-regulated in rice with limited nutrients (Cho et al., 2008). In A. thaliana, MCM3 is present in various oligomeric forms including as a homohexamer and possesses 3′ to 5′ helicase and ATPase activities in vitro (Rizvi et al., 2015). MCM7 protein is localized in the nucleus and is required for DNA replication and cytokinesis at an early stage of Arabidopsis development (Springer et al., 2000; Holding and Springer, 2002). The repression of MCM2, MCM3, MCM5, and MCM7 was associated with viral infection previously (Choi et al., 2015), but detailed functional studies have not been carried out. The down-regulation of photosynthetic capability, tetrapyrrole and lipid metabolism and DNA replication are all associated with iron deficiency. Similar symptoms of leaf yellowing following disturbances in heavy metal metabolism are found in citrus suffering from huanglongbing (HLB). In the case of HLB, foliar sprays of nutritional supplements alleviate symptoms (Spann and Schumann, 2009; Stansly et al., 2013), and may be worthwhile in the case of severe CTV as well.

Reduction of shoot growth and cell proliferation were found in ribosomal protein mutants (Byrne, 2009; Horiguchi et al., 2011, 2012). Many chloroplast ribosomal protein genes were strongly down-regulated in a related study in response to co-infection with CTV-B6 and “Candidatus Liberibacter asiaticus,” causal agent of citrus huanglongbing (Bové, 2006; submitted), and were also greatly repressed in response to CTV-B2/CTV-B6, including RPS17, RPL9, RPL17, and RPL18 (Table 2). Gene expression in chloroplasts is primarily controlled through the regulation of translation, mediated by the ribosome. Great alterations of ribosomal composition were observed in leaves and roots of Arabidopsis in response to iron and phosphate deficiency (Rodríguez-Celma et al., 2013; Wang et al., 2013). Gene expression in chloroplasts was limited and further affected the photosynthesis capacity.

Cellulose is the most abundant and strongest component of cell walls and it is targeted for degradation by pathogens to facilitate penetration of and movement between plant cells. Genes associated with cellulose synthase were induced by double infection with CTV-B2/CTV-B6 and by single infection with CTV-B2 but not by CTV-B6 alone (Fu et al., 2016). XTH gene expression and enzyme activity was positively associated with cell wall elongation (Shin et al., 2006) and strengthening (Antosiewicz et al., 1997). Expansin activity is most often in association with cell-wall loosening in growing cells (Lee et al., 2001), and expansin proteins are distributed throughout cell walls (Zhang and Hasenstein, 2000; Cosgrove et al., 2002). Both XTH and EXLB are also correlated with cell wall modification, which is an important aspect of plant responses to biotic and abiotic stresses, and is likely to be the relevant context in this study. The expression of EXLB3 was also up-regulated by single infection with CTV-B2 (Table 4), but not as strongly as in response to double infection with CTV-B2/CTV-B6. The stronger up-regulation of EXLB3 may be stimulated by the presence of virulent CTV-B6. The up-regulation of XTH and EXLB gene expression when under virus attack can modulate cell wall growth and equip the plant with a stronger physical barrier to deter feeding by the vector aphids. The remodeling and thickening of cell walls is also correlated with the symptoms of stiff and thickened leaves and vein corking (Figure 2).

Induction of transporters was also observed in a previous study (Fu et al., 2016). The up-regulation of sugar, amino acid, sulfate, phosphate, and metal and oligopeptide transporters denoted a shortage of these substances and a disturbance of the transportation system in young leaves prior to the development of symptoms. SUC2 (sucrose-proton symporter 2), encoding a sucrose/proton symporter was up-regulated and is necessary for phloem loading and is also fundamental for phloem transport and plant productivity (Srivastava et al., 2009).

Ammonium is the preferred form of nitrogen for uptake by plants (Gazzarrini et al., 1999) as it requires less energy for assimilation into amino acids (Bloom et al., 1992). The assimilation of ammonium from the soil solution is regulated by ammonium transporters (AMT) and AtAMT1;1 is expressed in roots and leaves (Engineer and Kranz, 2007).

The differential down-regulation level of phloem proteins in response to co-infection with CTV-B2/CTV-B6 and single infection with CTV-B2 are consistent with the restructuring of phloem, as well as with cell-to-cell and long distance movement of viruses (Imlau et al., 1999; Gómez and Pallás, 2004).

The up-regulation of ZIP11 was also observed in response to single infections with CTV-B2 or CTV-B6 (Table 4; Fu et al., 2016). However, the expression of ZIP5, as well as ZIP1, ZIP4, and was down-regulated by CTV-B2 in a single infection (Table 4). Zinc is a key element possessing both structural and functional roles in plants and about 4% of all predicted proteins in Arabidopsis contain one or more zinc fingers (Kawagashira et al., 2001). A large number of enzymes contain zinc binding sites, such as, copper-zinc superoxide dismutase (Hacisalihoglu et al., 2003) and zinc metallo proteases (Ståhl et al., 2002). The differential expression patterns of ZIP1, ZIP4, and ZIP5 in response to co-infection by CTV-B2/CTV-B6 and infection by CTV-B2 alone is probably due to severe, virulent CTV-B6, which affects the plant profoundly, inducing chlorosis of leaves, collapse of phloem and root decline.

Iron is also an essential metal nutrient required for a number of critical cellular functions such as, the synthesis of heme and heme-dependent oxygen transport, iron-dependent enzymatic reactions, photosynthesis and ribonucleotide synthesis (Straus, 1994; Kobayashi and Nishizawa, 2012). FRO6 (ferric reduction oxidase 6) was up-regulated in response to CTV-B2/CTV-B6 infection. FRO6 is involved in metal binding/acquisition and is responsible for regulating the reduction of iron from ferric to ferrous at the plasma membrane of leaf cells in response to iron deficiency (Maynes, 2013). Compared to roots of iron-sufficient plants, very high concentrations of manganese, zinc and copper accumulated in roots of iron-deficient Arabidopsis plants (Korshunova et al., 1999; Connolly et al., 2002; Vert et al., 2002). Hence, the high up-regulation of zinc transporters may be a secondary effect of iron deficiency.

Auxin regulates gene expression and facilitates elongation of shoot cells by activating the auxin response factor (ARF) family of transcription factors. Auxin is perceived by TIR1/AFB (transport inhibitor response1/auxin signaling F-BOX) family of F-box proteins (Salehin et al., 2015). In the presence of auxin, members of TIR1/AFB family of proteins regulate the polyubiquitylation and proteasomal degradation of Aux/IAA transcriptional repressors (Dharmasiri et al., 2005; Greenham et al., 2011). ARFs are repressed by the Aux/IAA proteins. Some of the most common symptoms produced by plant viruses are leaf curling, chlorosis, and stunting, are related to disruptions of plant hormone production, accumulation, and sensing (Jameson and Clarke, 2002; Padmanabhan et al., 2005). The auxin system has been directly disrupted by viral components in other systems. Auxin signaling was disrupted by the Tobacco mosaic virus (TMV) 126 KDa replication protein via an interaction with select Aux/IAA family members (Padmanabhan et al., 2005, 2006), which are removed by auxin-induced degradation. The up regulation of AFB5 and down regulation of ARF18 and ARF19 are signs of alterations in auxin metabolism, which may be associated with disease symptoms such as, stunting and leaf curling.

Ethylene is a vital growth regulator and is also important for biotic and abiotic stress responses (Schaller, 2012). Ethylene is perceived by a family of five membrane-associated receptors (ETR1, ETR2, ERS1, ERS2, and EIN4). The ethylene receptors are involved in transcriptional as well as post-transcriptional regulatory mechanisms (O'Malley et al., 2005; Chen et al., 2007; Kevany et al., 2007). The ethylene receptors function as negative regulators of ethylene responses and their function is inactivated through ethylene binding (Bleecker and Kende, 2000; Chang and Stadler, 2001). The up regulation of ERS1 and EIN4 imply enhanced ethylene production, which is an early and active response of plants to virus attack in association with the induction of defense responses (Boller, 1991).

Gibberellins are master regulators for plant growth in response to both abiotic (temperature, salt, and light) and biotic stress (Claeys et al., 2014; Colebrook et al., 2014). A giberellin-responsive pathway regulates photosynthesis (Xie et al., 2016). The up-regulation of AFB5, auxin-responsive protein, ERS1 and EIN4, and gibberellin-responsive protein are changes of hormone signaling pathways in plants under pathogen stress and lead to the induction of the following defense actions.

Terpenoids are a structurally diverse group of plant secondary metabolites and are involved in both direct and indirect plant defenses. Terpene synthases (TPSs) play an important role in the synthesis of volatile terpenes. The expression of TPS21 can be induced by gibberellin and jasmonate and the induction of TPS21 increases the emission of sesquiterpenes. The plant cuticle plays a vital role in the interactions between the plant and environment stresses, including water stress. Alkanes are prominent components of cuticular wax and the alkane-forming pathway is controlled by the co-expression of CER1/CER3 (eceriferum; Bernard et al., 2012). The increased expression of CER1/CER3 increases the amount of wax on the surface of the leaves. It is probable that increasing expression of these genes in association with hormone and signaling pathways mediated by secondary metabolites enhanced the tolerance of the plants to water stress and may deter feeding by the aphid vector of CTV.

When mild CTV-B2 and severe CTV-B6 were inoculated simultaneously, both mild and severe CTV strains successfully established in citrus plants. The sweet orange plants co-infected with both CTV strains showed symptoms typical of CTV-B6, indicating virulent CTV-B6 is the dominant strain and that neither virus affected the other's establishment in sweet orange. The failure of CTV-B2 to cross protect is likely because the two virus strains are from different genotypes (Folimonova et al., 2010; Folimonova, 2013). The photosynthetic and carbon metabolism capacities of host plants were greatly reduced, and the composition of the ribosome was greatly altered in response to co-infection. These responses may be attributed to deficiency of iron, and secondarily to zinc deficiency. Host plant metal ion transport systems were generally disturbed and ZIPs showed very different expression patterns in response to co-infection by CTV-B2/CTV-B6 and to single strain infection by CTV-B2, which was due to the presence of virulent CTV-B6.

The outcome of a plant–virus interaction from antagonism to mutualism is determined by the environment, host genotype, and pathogens. Virulence is the negative impact of parasite infection on host fitness (Read, 1994; Little et al., 2010) and is determined by both the host and pathogen. Host defenses may decrease virulence through either resistance or tolerance. The former limits the multiplication of the parasite and the latter decreases damage to the host in spite of the amount of multiplication by the pathogen. C. sinensis is generally susceptible to CTV. Though defense responses, such as, strengthening of cell walls, altered hormone metabolism, increased production of secondary metabolites and signaling proteins were induced, these did not suppress the spread of the pathogens and the development of symptoms caused by CTV-B2/CTV-B6, or strain CTV-B6 alone, although they were able to induce tolerance to strain CTV-B2 (Fu et al., 2016).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.