Abstract

   Citrus Huanglongbing (HLB), a highly devastating citrus disease, is
   associated with ‘Candidatus Liberibacter asiacitus’ (CLas), a member of
   phloem-inhabiting α-proteobacteria. HLB can affect all cultivated
   citrus and no cure is currently available. Previous studies showed that
   Kaffir lime (Citrus hystrix), primarily grown in South Asia and
   Southeast Asia, was tolerant to HLB but the molecular mechanism remains
   unknown. In this study, gene expression profiling experiments were
   performed on HLB-tolerant C. hystrix and HLB-susceptible C. sinensis
   three months after inoculation with CLas using RNA-seq data.
   Differentially expressed genes (DEGs) in the two citrus cultivars were
   mainly involved in diverse cellular functions including carbohydrate
   metabolism, photosynthesis, cell wall metabolism, secondary metabolism,
   hormone metabolism and oxidation/reduction processes. Notably, starch
   synthesis and photosynthesis process were not disturbed in
   CLas-infected C. hystrix. Most of the DEGs involved in cell wall
   metabolism and secondary metabolism were up-regulated in C. hystrix. In
   addition, the activation of peroxidases, Cu/Zn-SOD and POD4, may also
   enhance the tolerance of C. hystrix to CLas. This study provides an
   insight into the host response of HLB-tolerant citrus cultivar to CLas.
   C. hystrix is potentially useful for HLB-tolerant/resistant citrus
   breeding in the future.

Introduction

   Citrus is an important economic crop in tropical and sub-tropical
   regions worldwide. Global citrus production in 2014 exceeded 121
   million metric tons
   ([34]http://www.fao.org/economic/est/est-commodities/citrus/en/),
   ranked the top among all the fruit crops. Citrus Huanglongbing (HLB,
   yellow shoot disease, also known as citrus greening disease) is one of
   the most destructive diseases in citrus production [[35]1]. The disease
   is associated with three species of non-culturable, phloem-limited,
   α-Proteobacteria: ‘Candidatus Liberibacter asiaticus’, ‘Ca. L.
   africanus’, and ‘Ca. L. americanus’ [[36]2, [37]3]. Among the three
   species, ‘Ca. L. asiaticus’ (CLas) is the most prevalent in major
   citrus-producing regions, e.g. China, Brazil and the United States.

   No effective cure is currently available for HLB-infected citrus
   plants. Integrated management practices including the use of
   pathogen-free nursery stocks, control of insect vectors and removal of
   infected trees are the commonly recommended strategies. Although all
   commercial citrus cultivars are susceptible to HLB, a few HLB
   resistance or tolerance citrus and citrus relativeshave been reported
   [[38]4–[39]6]. Kaffir lime (C. hystrix), primarily grown in South and
   Southeast Asia, has been used for cooking and traditional medicine.
   Extracts of Kaffir lime leaves, rich in terpenoids and phenolic
   contents, could inhibit the proliferation of cancer cells and biofilm
   formation of bacteria [[40]7, [41]8]. C. hystrix was experimentally
   validated as HLB-tolerance citrus cultivar in Malaysia [[42]4, [43]9].
   Similar result was also obtained in our laboratory based on three-year
   biological indexing and qPCR monitoring (unpublished data).

   To better understand the HLB resistance and tolerance mechanisms in
   citrus, substantial research efforts have been made to study
   citrus-CLas interaction through microarray and high-throughput
   sequencing technology [[44]10–[45]16]. Gene expression profiles of
   different citrus leaves, fruits and roots from susceptible citrus
   cultivars infected with CLas showed that genes involved in sugar and
   starch metabolism, photosynthesis, cell wall metabolism, stress
   response and hormone signaling were significantly altered
   [[46]10–[47]16]. Comparative transcriptional and transcriptome studies
   between HLB tolerant and susceptible citrus cultivars were performed to
   identify genes of HLB tolerance to HLB (17–19). The HLB tolerance
   breeding line US-897 (C. reticulata×P. trifoliata) might be associated
   with the constitutively higher expression of defense-related genes,
   compared with the susceptible ‘Cleopatra’ mandarin (C. reticulata)
   [[48]17]. Comparative transcriptional analysis of tolerant rough lemon
   (C. jambhiri) and susceptible sweet orange (C. sinensis) in response to
   CLas infection revealed more differentially expressed genes in
   HLB-infected rough lemon than those in sweet orange at early stages but
   substantially fewer at late time points. Genes coding for cell wall
   proteins, β-1, 3-glucanases, and GASA1, were identified as potential
   HLB tolerant citrus improvements program [[49]18]. Recently, expression
   differences in secondary metabolites, pathogenesis related genes,
   transcription factors, hormone signaling pathways and receptor-like
   kinases were found from ‘Jackson’ grapefruit-like-hybrid and
   susceptible ‘Marsh’ grapefruit (C. paradisi) [[50]19].

   In light of the successful examples above, this study was to study
   transcriptical difference between highly susceptible sweet orange trees
   and tolerant kaffir lime trees using RNA-seq approach. The
   identification of differentially expressed genes in the two citrus
   cultivars at an early stage of HLB infection provides new information
   for future HLB management.

Materials and methods

Plant materials

   The disease free budwoods of tolerant kaffir lime (C. hystrix) and
   susceptible pineapple sweet orange (C. sinensis) collected from
   virus-free citrus repository of National Citrus Virus Exclusion Center
   (NCVEC) in Southwest University were grafted on two-year-old Carrizo
   citrange rootstocks (C. sinensis×P. trifoliata), respectively. The
   budwood used for inoculation was Guanximiyou pummelo (C. grandis)
   solely infected with CLas strain GZBJT maintained in greenhouse in
   NCVEC. Three replicate trees per genotype were infected with CLas
   through bud grafting, with three CLas-free bud grafted trees as the
   control.

DNA extraction and ‘Ca. L. asiaticus’ detection

   Fully expanded mature leaves were collected from diseased and
   mock-inoculated trees every twenty days for DNA extraction described
   previously [[51]20]. Both conventional PCR using primer set OI1/OI2c
   and TaqMan qPCR assays with an iCycler IQ5 (BioRad, Hercules, CA) were
   used to detect CLas [[52]2, [53]21].

RNA extraction and RNA-sequencing

   Once infection had been confirmed by PCR and qPCR, total RNA were
   extracted from fully expanded young leaves with mortar and pestle in
   liquid nitrogen. RNA extraction process was performed according to
   TRIzol protocol (Invitrogen, Carlsbad, CA). The concentration of total
   RNA was determined by Nanodrop 2000 Spectrophotometer, and the RNA
   integrity number (RIN) was evaluated by Agilent 2100 Bioanalyzer. Three
   RNA replicates of each treatment were pooled in equal amount, and sent
   to Beijing Genomics Institute (BGI)-Wuhan (China) for RNA sequencing.

Data analysis

   After cleaning the raw reads, we mapped the clean reads to Citrus
   sinensis genome ([54]http://www.ncbi.nlm.nih.gov/genome/ 10702) using
   Bowtie 2 [[55]22] and then calculated gene expression level with RSEM
   [[56]23]. Differentially expressed genes (DEGs) were screened based on
   PossionDis with a cutoff threshold FDR of 0.001 and log[2] fold change
   (FC) of ≥ 1.00 or ≤ -1.00.

   Functional enrichment analysis was carried out using the GO database
   ([57]http://www.geneontology.org/). The DEGs were also analyzed by
   PageMan [[58]24] embedded in MapMan [[59]25]. A Wilcoxon test was
   applied and a statistics-based overview of changed pathways from global
   gene expression alterations was provided. The same data sets were also
   imported into MapMan to map the differentially expressed genes into
   specific pathways.

RT-qPCR validation

   To verify the results of RNA-seq, 16 DEGs involved in carbohydrate
   metabolism, photosynthesis, cell wall metabolism, secondary metabolism,
   hormone metabolism and Pathogenesis related (PR)-proteins were selected
   for RT-qPCR analysis. The same batch RNA samples used in RNA-seq
   analysis were used for RT-qPCR. The first strand cDNA was synthesized
   using HiScript^® Reverse Transcriptase (Vazyme, Nanjing, China) and 6N
   random primers according to the manufacture’s protocol. RT-qPCR was
   performed on an iCycler IQ5 instrument with SYBR^® Premix Ex Taq^™ II
   (TakaRa, Dalian, China). The cycling conditions included incubation for
   30 s at 95°C followed by 40 cycles of amplification (95°C for 5 s and
   60°C for 25 s). GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was
   selected as the internal reference gene [[60]26] and the relative
   expression values were calculated with Ct method (2^-ΔΔCt). The primers
   used in RT-qPCR are listed in [61]S1 Table.

Results and discussion

Identification of differentially expressed genes

   Around 44 million clean reads were obtained from infected and control
   plants and mapped to Citrus sinensis genome [[62]27], with
   approximately 62% success for C. hystrix, and 77%-78% for C. sinensis
   libraries ([63]S2 Table). Dramatic differences between the
   transcriptome profiles of C. hystrix and C. sinensis were observed in
   response to CLas infection. Overall, the number of differentially
   expressed genes (DEGs) was greater in susceptible sweet orange than in
   tolerant kaffir lime when compared to their respective healthy control.
   Among these DEGs, 179 genes were up-regulated and 73 were
   down-regulated in C. hystrix, while 254 genes were up-regulated and 350
   were down-regulated in C. sinensis ([64]Fig 1). Due to the differences
   in genetic background between the two citrus cultivars, the DEG number
   of infected/healthy C. hystrix in relation to infected/healthy C.
   sinensis was much greater than the number of the two infected cultivars
   in relation to their mock-inoculated healthy controls ([65]Fig 1).

Fig 1. Statistic of differentially expressd genes (DEGs) of different citrus
cultivars in response to CLas.

   [66]Fig 1
   [67]Open in a new tab

   CH, Citrus hystrix; CS, C. sinensis; M, Mock/healthy; HLB,
   Huanglongbing; CH-M-VS-CH-HLB, DEGs in HLB-infected C. hystrix compared
   with healthy control; CS-M-VS-CS-HLB, DEGs in HLB-infected C. sinensis
   compared with healthy control; CS-M-VS-CH-M, DEGs between healthy C.
   hystrix and healthy C. sinensis; CS-HLB-VS-CH-HLB, DEGs between
   HLB-infected C. hystrix and HLB-infected C. sinensis.

Gene ontology classification of DEGs

   The DEGs were enriched into different function categories through Gene
   ontology (GO) enrichment analysis ([68]Fig 2). Significantly enriched
   GO terms were extracted using TopGO [[69]28]. For DEGs in HLB tolerant
   C. hystrix, the most significantly enriched GO terms in biological
   process were related to ‘threonine metabolic process’ (GO:0006566),
   ‘negative regulation of peptidase activity’ (GO:0010466), ‘regulation
   of proteolysis’ (GO:0045861), and ‘regulation of peptidase activity’
   (GO:0052547). The most significant GO terms in molecular function
   included ‘terpene synthase activity’ (GO: 0010333), and ‘carbon-oxygen
   lyase activity, acting on phosphates’ (GO:0016838). No GO term in
   cellular component was enriched in DEGs in C. hystrix ([70]S2 Table).
   For DEGs in susceptible C. sinensis, only two GO terms in biological
   process, ‘reactive oxygen species metabolic process’ (GO: 0072593) and
   ‘cellular protein complex assembly’ (GO: 0043623) were enriched. The
   most significant GO terms in molecular function were related to
   ‘transferase activity’ (GO: 0016758), ‘cellulose synthase activity’
   (GO: 0016759), and ‘glucuronosyltransferase activity’ (GO: 0015020).
   Only three GO terms in cellular component were enriched in DEGs in C.
   sinensis and the most significant one was ‘external encapsulating
   structure’ (GO: 0030312) ([71]S3 Table).

Fig 2. Gene ontology classification of differentially expressed genes in
different citrus hosts in response to ‘Candidatus Liberibacter asiaticus’.

   [72]Fig 2
   [73]Open in a new tab

   BP, Biological Process; CC, Cell Component; MF, Molecular Function.

   DEGs within two infected cultivars were mainly enriched in nine
   biological process GO terms, including ‘drug transport’ (GO: 0015893),
   ‘response to drug’ (GO: 0042493) and ‘response to chemical’
   (GO:0042221). Besides, nine molecular function GO terms were enriched,
   such as ‘catalytic activity’ (GO: 0003824), ‘oxidoreductase activity’
   (GO: 0016491), ‘transferase activity’ (GO: 0016758, GO: 0016757),
   whereas only one cellular component GO term, ‘intrinsic component of
   membrane’ (GO: 0031224), was enriched in DEGs in the two cultivars in
   response to CLas ([74]S4 Table).

Gene pathway enrichment analysis of CLas-modulated host pathways

   PageMan analysis showed that only three pathways significantly enriched
   were associated with the DEGs identified in C. hystrix ([75]Fig 3).
   Pathways related to major carbohydrates (CHO) metabolism was
   up-regulated, while pathways related to posttranslational modification
   and LRR XII receptor kinases were down-regulated in tolerant citrus
   plants. Instead, much more pathways were significantly enriched in C.
   sinensis ([76]Fig 3). The up-regulated pathways were starch synthesis,
   cell wall degradation, stress biotic, regulation of transcription,
   receptor kinases signaling, and metal transport in susceptible citrus
   plants. The down-regulated pathways included cellulose synthesis,
   hemicellulose synthesis, invertase/pectin methylesterase inhibitor
   family protein, GDSL motif lipase, and aspartate protease mediated
   protein degradation.

Fig 3. Comparative PageMan display of perturbed pathways in CLas-affected C.
hystrix and C. sinensis.

   [77]Fig 3
   [78]Open in a new tab

   The log[2] fold change of gene expression (mock-inoculated controls
   versus CLas-inoculated plants) was input into PageMan and subjected to
   a Wilcoxon test. Results were shown as a false-color heat-map-like
   display. Significantly up-regulated pathways are colored in red, while
   those colored in green are significantly down-regulated. Pathways
   without significant changes are white. Names of pathways are indicated
   on the right panel. CH, CH-M-VS-CH-HLB; CS, CS-M-VS-CS-HLB.

   MapMan software was applied to display and analyze the functional
   classes those were significantly different in CLas-affected C. hystrix
   and C. sinensis. The results showed that DEGs were mainly involved in
   diverse cellular functions including carbohydrate metabolism,
   photosynthesis, cell wall metabolism, secondary metabolism, hormone
   metabolism, PR proteins and oxidation/reduction processes (Figs [79]4
   and [80]5, [81]S5 Table).

Fig 4. Differentially expressed genes involved in starch metabolism (A) and
photosynthesis (B).

   [82]Fig 4
   [83]Open in a new tab

   Colored squares indicate up- or down-regulated genes with log[2] fold
   change (FC) ≥ 1.00 or ≤ -1.00.

Fig 5. MapMan analysis of differentially expressed genes in CLas-affected
Citrus hystrix (A) and CLas-affected C. sinensis (B) involved in stress
responses.

   [84]Fig 5
   [85]Open in a new tab

   Red squares represent genes those were significantly up-regulated;
   green squares represent genes those were significantly down-regulated.

Carbohydrate metabolism and photosynthesis process

   Cytopathology in different stage of HLB infection revealed swelling of
   the middle lamella between cell walls surrounding sieve elements in
   non-symptomatic citrus new flushes at the early infection stage and
   then necrosis of sieve elements and companion cells and phloem plugging
   by the callose-like material and excessive starch at the later
   infection stage [[86]29–[87]31]. It suggests that the disturbance of
   starch metabolism and the inhibition of the transport of photosynthate
   may contribute to HLB development in citrus hosts. Microarray or
   RNA-seq analysis also proved that carbohydrate metabolic process was
   significantly changed and photosynthesis was repressed in HLB-affected
   citrus [[88]15, [89]17, [90]18, [91]30, [92]32]. From these studies,
   the key starch synthesis genes were generally up-regulated and the
   starch degrading genes were down-regulated in affected susceptible
   citrus. Recently, proteomic analysis suggested no clear correlation was
   observed from starch pathway regulation between moderately tolerant
   cultivar ‘Volkameriana’ and susceptible navel orange [[93]33].

   In this study, several genes involved in carbohydrate metabolism were
   regulated in the early stage of infection with CLas ([94]Fig 4, [95]S5
   Table). The starch synthesis was induced while the starch degradation
   was depressed in C. sinensis. Two glycogen synthase (starch synthase,
   glgA) geneswere significantly up-regulated, whereas beta-amylase (BAM)
   gene was down-regulated in C. sinensis ([96]Fig 4, [97]S5 Table).
   However, the expression levels of these genes were not significantly
   changed in CLas-affected C. hystrix. Instead, one gene encoding
   alpha-amylase (AAM), associated with starch degradation, was slightly
   up-regulated in C. hystrix ([98]Fig 4, [99]S5 Table).

   Genes encoding light-harvesting complex II chlorophyll a/b binding
   protein 1, Photosystem II psbP domain-containing protein 1 and
   photosystem I subunit O, which involved in light action of
   photosynthesis, were all down-regulated in C. sinensis ([100]Fig 4,
   [101]S5 Table). The three genes were not significantly changed in C.
   hystrix, suggesting the photosynthesis process may not be depressed in
   tolerant C. hystrix.

   Taken together these findings showed that the repression of key
   proteins involved in photosynthetic light reactions and up-regulation
   of starch-related pathways in C. sinensis was in agreement with
   previous studies. Notably, no significant change in starch synthesis
   and photosynthesis process may play important roles in tolerance of C.
   hystrix. Anatomical evidences from different infection stage of kaffir
   limeare required to prove the transcriptome data.

Genes involved in cell wall and cell organization

   The plant cell wall is comparable to an exoskeleton surrounding the
   plant cell and providing both structural support and protection from
   biotic as well as abiotic stresses [[102]34]. It is well established
   that the plant susceptibility to pathogens depends on the cell wall
   composition and structure, which determines its recalcitrance to
   degradation by cell wall modifying enzymes (CWMEs) produced by
   pathogens [[103]35]. Plant cell walls are composed of layers of
   cellulose microfibers embedded in a matrix of pectin and hemicellulose,
   plus some structural proteins [[104]36–[105]38]. There were 44 and 13
   genes found related to cell wall metabolism among the DEGs in
   HLB-susceptible and tolerant cultivars, respectively ([106]Fig 5,
   [107]S5 Table). Overall, most genes involved in cellulose synthesis,
   cell wall proteins and cell wall modification were depressed in
   diseased C. sinensis. For instance, several genes encoding expansin,
   which are related to cell wall breakdown, including EXLA1, EXPA5 and
   EXLB1, were down-regulated in C. sinensis, and EXLA1 was also
   down-regulated in C. hystrix. Expansin related genes were reported to
   exhibit high expressions in susceptible ‘Marsh’ grapefruit [[108]19].
   Down-regulation of these genes in both C. hystrix and C. sinensis
   indicates a host defense response to CLas infection. A group of genes
   encoding xyloglucan endotransglucosylase/hydrolase proteins (XTH22,
   XTH23), associated with cell wall degradation, were slightly
   up-expressed in CLas-affected C. sinensis, whereas no significant
   difference was observed in CLas-affected C. hystrix. Furthermore, DEGs
   involved in cellulose synthesis, such as cellulose synthase A (CESA),
   cellulose synthase-like protein A2 (CSLA2), cellulose synthase-like
   protein A9 (CSLA9) and cellulose synthase-like protein C5 (CSLC5) were
   all down-regulated in CLas-affected C. sinensis, while CSLA2 and CSLA9
   were up-regulated in CLas-affected C. hystrix. Thus, kaffir lime
   responded to CLas by activating enzymes that would work together to
   strengthen the cell wall, which may contribute to the reinforcement of
   physical barriers to restrict the invasion of CLas.

Secondary metabolism

   Secondary metabolism plays important roles in plant defenses. Previous
   studies revealed that most genes involved in secondary metabolites,
   including terpenoid, flavonoid and phenylpropanoid biosynthesis
   pathways, were highly induced in CLas-affected leaves [[109]15,
   [110]17, [111]19, [112]39], whereas most of these genes were
   down-regulated in roots following HLB infection [[113]16]. Here, most
   DEGs involved in secondary metabolism pathway were activated in C.
   hystrix, but were suppressed in C. sinensis. These DEGs were
   categorized into the biological processes of terpene biosynthesis,
   flavonoids biosynthesis and phenylpropanoids biosynthesis ([114]Fig 5,
   [115]S5 Table). Terpenoids are a structurally diverse group of natural
   products, which function as plant antioxidants, insect attractants or
   repellents [[116]40, [117]41]. Flavonoids are anti-fungi substances and
   antioxidants [[118]42]. Phenylpropanoids serve as structural polymers
   including lignins, provide protection from pests and UV light and
   attract pollinators as pigments [[119]43]. Two genes encoding
   germacrene D synthase-like (GDSL) were up-regulated in both two
   cultivars in response to CLas infection. Two genes encoding
   gamma-terpinene synthase (TPS2) were only up-regulated in HLB tolerant
   citrus trees. The expression level of one gene encoding (R)-limonene
   synthase 1 was increased in C. hystrix, while the other two genes
   related to this enzyme were suppressed in C. sinensis. GDSL, TPS2 and
   (R)-limonene synthase were involved in terpene biosynthesis. One gene
   encoding 2-oxoglutarate and Fe (II)-dependent (2OG-Fe (II)) oxygenases,
   which is associated with flavonoids biosynthesis, was strongly
   up-regulated in C. hystrix, whereas it was down-regulated in C.
   sinensis. Genes involved in phenylpropanoids biosynthesis such as
   isoflavone reductase (IRL) and cinnamoyl-CoA reductase 1 (CCR), were
   induced in C. hystrix, while phenylalanine ammonia-lyase (PAL) and
   4-coumarate—CoA ligase 1 (4CL1), which also play an important role in
   phenylpropanoids biosynthesis, were suppressed in C. sinensis.

   The tolerant cultivars contain relatively higher specific volatile
   compounds such as monoterpenes and aldehydes than susceptible cultivars
   [[120]44]. It should be noted that Kaffir lime leaves contain various
   classes of phytochemical substances including terpenoids and
   polyphenolic which are known for their antimicrobial activities
   [[121]7, [122]8]. The up-regulation of genes involved in secondary
   metabolism pathways, and high phytochemical substances containing in
   kaffir lime leaves may contribute to its tolerance to CLas.

Hormone metabolism

   Phytohormones play critical roles in helping the plants to adapt to
   adverse environmental conditions. The elaborate hormone signaling
   networks and their ability of crosstalk make them ideal candidates for
   mediating defense responses [[123]45]. There were 35 hormone signaling
   genes in identified DEGs ([124]Fig 5, [125]S5 Table). Auxin was found
   to promote the expression of expansins in plants [[126]46–[127]48],
   which contribute to breakage of plant cell walls. Of the seven
   auxin-related genes, three genes related to crocetin
   glucosyltransferase were up-regulated in C. hystrix, two
   auxin-responsive proteins, SAUR72 and IAA1, were down-regulated in
   C.sinensis. SAUR was proved to act as a negative regulator of auxin
   synthesis in rice and may promote resistance to pathogens [[128]49].
   Transcription analysis showed that three SAUR-like genes were
   up-expressed in HLB tolerant ‘Jackson’ grapefruit, which may also
   contribute to the down expression of expansin [[129]19]. A gene
   encoding the senescence-related gene (SRG1), involved in ethylene
   metabolism, was highly induced in response to CLas in C. hystrix. The
   expression of ethylene-responsive transcription factor ERF07 was
   up-regulated in both cultivars, while ERF107 was down-regulated.
   Ethylene-responsive transcription factor and EIN3-binding F-box protein
   1 were only up-regulated in C. sinensis. Notably, five genes involved
   in salicylic acid metabolism were suppressed in C. sinensis but not in
   C. hystrix. JA levels in response to pathogen infection clearly
   highlight its involvement in plant defense responses. The key enzyme
   involved in jasmonate synthesis, lipoxygenase 2 (LOX2) [[130]50], were
   up-regulated in both cultivars. JA-responsive gene expression for
   defense response is mainly mediated by a transcription factor JASMONATE
   INSENSITIVE 1/MYC2 (JIN1/MYC2) [[131]51]. Up-regulation of
   transcription factor MYC2 was only observed in C. hystrix. These
   results showed that JA signal transduction may be activated by
   infection with CLas, especially in C. hystrix.

Pathogenesis-related (PR) genes

   The pathogenesis-related (PR) proteins of plants are a group of
   host-encoded, inducible proteins whose synthesis is often associated
   with certain forms of resistance to pathogens and stresses [[132]52].
   Previous studies showed that most genes related to PR-proteins were
   induced by CLas infection, including receptor-like protein kinase,
   TIR-NBS-LRR (Toll interleukin-1 receptor nucleotide-binding site
   leucine-rich repeat) class and NB-ARC (nucleotide binding-adaptor
   shared by APAF-1, certain R gene products and CED4) domain proteins,
   and Kunitz family trypsin and protease inhibitor protein [[133]11,
   [134]18, [135]32, [136]39].

   In this work, more genes related to PR genes were induced in C.
   sinensis than in C. hystrix ([137]Fig 5, [138]S5 Table). Several genes
   related to kinases involved in biotic stress, including LRR
   receptor-like serine/threonine-protein kinase GSO1, LRR receptor-like
   serine/threonine-protein kinase ERL2, LRR receptor-like protein kinase
   At1g35710, and receptor-like protein 12, were slightly up-expressed in
   C. sinensis. A gene encoding leucine-rich repeat receptor-like
   serine/threonine-protein kinase (At2g24130) was up-regulated more than
   8-fold in C. hystrix, but showed no significant difference in C.
   sinensis. Notably, two genes encoding miraculin, a Kunitz family
   trypsin and protease inhibitor protein which are associated with plant
   defenses, were only up-regulated in C. hystrix ([139]S5 Table). In
   addition, some genes related to disease resistance proteins, such as
   RPS4 and RPM1, exhibited higher expression level in C. hystrix. RPS4
   and RPM1 act as R genes to participate in the effector-triggered
   immunity (ETI) process, which play an important role in plant defense
   response [[140]53, [141]54].

Oxidation/Reduction processes

   14 genes involved in oxidation/reduction processes were differentially
   regulated between the HLB-susceptible and tolerant cultivars. It should
   be noted that most of them were up-regulated in tolerant C. hystrix and
   down-regulated in C. sinensis. Up-regulation of several glutaredoxin
   genes such as GRXC6, GRXC9 and GRXS9, were only identified in C.
   hystrix. These proteins have activity of glutathione-disulfide bond
   oxidordeuctase, which can reduce the amount of micromolecular disulfide
   and proteins. A gene encoding thioredoxin M3, a thiol-disulfide bond
   oxidordeuctase which required for maintaining permeability of plant
   meristem and regulation of callose deposition in Arabidopsis, was
   inhibited in C. sinensis. Besides, a group of superoxide dismutase
   (SOD) and peroxidase (POD) genes were differentially expressed in both
   cultivars. Of them, Cu/Zn-SOD and POD4 were up-regulated in C. hystrix,
   whereas they were down-regulated in C. sinensis ([142]S5 Table).
   Proteins involved in oxidation/reduction processes, including
   peroxidases and glutathione-S-transferases, are usually associated with
   the prevention of oxidative stress, which are induced by reactive
   oxygen species (ROS). Proteomic studies revealed that a higher
   activation of glutathione-S-transferases, which include several
   isozymes that help detoxify xenobiotic compounds, was observed in
   tolerant cultivar ‘Volkameriana’ [[143]33]. Therefore, activation of
   peroxidases, Cu/Zn-SOD and POD4, may also enhance the tolerance of C.
   hystrix to CLas.

RT-qPCR validation

   To validate the accuracy of the RNA-seq data, 16 genes classified in
   different function groups were tested by RT-qPCR. The expression
   profiles of 15 genes were consistent with the RNA-seq data,
   demonstrating the reliability of RNA-seq analysis. The RT-qPCR result
   of LHCb, encoding light-harvesting complex II chlorophyll a/b binding
   protein 1, did not agree with those obtained from the RNA-seq in C.
   hystrix. In addition, several genes showed no difference in RNA-seq
   data, but differentially expressed by RT-qPCR analysis. For example,
   glgA and XTH23 showed lower expression levels, PAL and RGA3 showed
   higher expression levels in CLas-infected C. hystrix based on qRT-PCR
   analysis ([144]Fig 6).

Fig 6. RT-qPCR and RNA-seq profiles of 16 selected differentially expressed
genes (DEGs).

   [145]Fig 6
   [146]Open in a new tab

   A. DEGs between healthy and HLB-infected Citrus sinensis; B, DEGs
   between healthy and HLB-infected C. hystrix; C, DEGs between
   HLB-infected C. hystrix and HLB-infected C. sinensis. glgA, starch
   synthase; TPP, trehalose 6-phosphate phosphatase; LHCb1,
   light-harvesting complex II chlorophyll a/b binding protein 1; PAL,
   phenylalanine ammonia-lyase-like; 2OG-Fe(II), 2-oxoglutarate (2OG) and
   Fe(II)-dependent oxygenase; CESA, cellulose synthase A; XTH23,
   xyloglucan endotransglucosylase/hydrolase protein 23; PHT, phosphate
   transporter; ZIP2, zinc transporter 2; EREBP, ethylene-responsive
   transcription factor ERF017; SODC, superoxide dismutase [Cu-Zn]; LRR-1,
   LRR receptor-like serine/threonine-protein kinase; LRR-2, LRR-like
   serine/threonine-protein kinase BAM2; RGA3, disease resistance protein
   RGA3, NB-ARC class; EDS1 (enhanced disease susceptibility 1 protein);
   MIR, miraculin.

Conclusions

   Global transcriptome profiles between the HLB tolerant C. hystrix and
   susceptible C. sinensis showed that specific responses in carbohydrate
   metabolism, photosynthesis process, cell wall metabolism, secondary
   metabolism and oxidation/reduction processes may play important roles
   against CLas attack in C. hystrix. 1) starch synthesis and
   photosynthesis process were not disturbed in CLas infected C. hystrix.
   2) several cellulose synthase and cellulose synthase-like family
   proteins, which may strengthen the cell wall synthesis, were induced in
   C. hystrix. 3) most DEGs involved in secondary metabolism pathways were
   activated in C. hystrix, but were depressed in C. sinensis. 4) the
   activation of peroxidases, Cu/Zn-SOD and POD4, may also enhance the
   tolerance of C. hystrix to CLas, acting as detoxification proteins.

Supporting information

   S1 Table. The primers used for RT-qPCR validation.

   (XLS)
   [147]Click here for additional data file.^ (21.5KB, xls)
   S2 Table. Gene ontology enrichment results of differentially expressed
   genes in Citrus hystrix.

   (XLS)
   [148]Click here for additional data file.^ (27KB, xls)
   S3 Table. Gene ontology enrichment results of differentially expressed
   genes in Citrus sinensis.

   (XLS)
   [149]Click here for additional data file.^ (24.5KB, xls)
   S4 Table. Gene ontology enrichment results of differentially expressed
   genes between Citrus hystrix and C. sinensis.

   (XLS)
   [150]Click here for additional data file.^ (25.5KB, xls)
   S5 Table. Differentially expressed genes in Citrus hystrix and C.
   sinensis in response to infection by CLas.

   (XLS)
   [151]Click here for additional data file.^ (52KB, xls)

Acknowledgments