Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Phloem and Xylem Responses Are Both Implicated in Huanglongbing Tolerance of Sugar Belle.

Although huanglongbing (HLB) is a devastating citrus disease, improved tolerant cultivars, such as Sugar Belle (SB) mandarin, have been identified. To understand the responses that HLB-affected SB undergoes, we compared 14CO2 fixation, carbohydrate export, phloem callose accumulation, relative expression of plant defense activators, and anatomical changes between healthy and infected SB trees versus susceptible Pineapple (PA) sweet orange. Eight- to ten-week-old leaves of infected SB showed a 2.5-fold increase in 14CO2 fixation and a 13% decrease in 14C-carbohydrate export, whereas HLB-affected PA presented a decrease of 33 and 50%, respectively. The mean distance of a callose deposit to its closest neighbor was 36% smaller in infected SB versus healthy, whereas in HLB-affected PA, it was 33% higher. Expression of papain-like cysteine proteases (PLCPs) was upregulated in SB but downregulated in PA. Infected SB showed minor alterations in the number of xylem vessels, a 16% larger xylem vessel lumen area, and a 14% increase in the proportional area of the xylem. In contrast, PA showed a 2.4-fold increase in the xylem vessel number and a 2% increase in the proportional xylem area. Three complementary mechanisms of tolerance in SB are hypothesized: (i) increased carbohydrate availability induced by greater CO2 fixation, mild effect in carbohydrate export, and local accumulation of callose in the phloem; (ii) activation of defense response via upregulation of PLCPs, and (iii) increased investment in the xylem structure. Thus, phloem and xylem modifications seem to be involved in SB tolerance.

Crossing the Gateless Barriers: Factors Involved in the Movement of Circulative Bacteria Within Their Insect Vectors.

Plant bacterial pathogens transmitted by hemipteran vectors pose a large threat to the agricultural industry worldwide. Although virus-vector relationships have been widely investigated, a significant gap exists in our understanding of the molecular interactions between circulative bacteria and their insect vectors, mainly leafhoppers and psyllids. In this review, we will describe how these bacterial pathogens adhere, invade, and proliferate inside their insect vectors. We will also highlight the different transmission routes and molecular factors of phloem-limited bacteria that maintain an effective relationship with the insect host. Understanding the pathogen-vector relationship at the molecular level will help in the management of vector-borne bacterial diseases.

Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7ko plants is accompanied by enhanced expression of carbohydrate transporters.

MAIN CONCLUSION: Loss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport. Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant's response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Intracellular Life Cycle of 'Candidatus Liberibacter asiaticus' Inside Psyllid Gut Cells.

'Candidatus Liberibacter asiaticus' (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas-efficient propagation in the vector, the Asian citrus psyllid Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut, the major organ for CLas transmission, is fundamental to improving current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles, termed Liberibacter-containing vacuoles (LCVs), by endocytosis. Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). Additionally, we observed the formation of double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas uses endocytosis- and exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic, and exocytic pathways via complex machineries. Potential tactics for HLB control can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.

Transcriptome Profiling of 'Candidatus Liberibacter asiaticus' in Citrus and Psyllids.

'Candidatus Liberibacter asiaticus' (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp.

Transgenic 'Duncan' grapefruit (Citrus paradisi Macf.) and 'Valencia' sweet orange (Citrus sinensis [L.] Osbeck) plants ectopically expressing C. sinensis (cv. Washington navel orange) APETALA1 (CsAP1) or LEAFY (CsLFY) genes under control of the Arabidopsis thaliana stress-inducible promoter AtRD29A flowered under non-inductive (warm temperature, well-watered) greenhouse conditions, whereas their wild-type (WT) counterparts did not. The transgenic plants that flowered exhibited no altered morphological features, except the lack of thorns characteristic of juvenile WT plants. The most precocious T0 line, 'Duncan' grapefruit (Dun134-3) expressing the CsAP1 gene, flowered and fruited when it was 4.5 years old and the T1 siblings from this line flowered and fruited when they were just over 18 months old. In contrast, T1 seedlings from three lines of 'Duncan' grapefruit expressing the CsLFY gene flowered within 3 months after germination, but were unable to support fruit development. Transcript levels of corresponding transgenes in leaves were not correlated with earliness of flowering. To further study the activity of AtRD29A, leaves from three 'Carrizo' citrange (C. sinensis × Poncirus trifoliata) rootstock seedlings transformed with the green fluorescent protein (GFP) gene under regulation of the AtRD29A promoter were subjected to drought stress or well-watered conditions. Expression of GFP was not stress-dependent, consistent with the observation of flowering of CsAP1 and CsLFY transgenic plants under non-inductive conditions. Taken together, the results suggest that AtRD29A is constitutively expressed in a citrus background. Despite the loss of control over flowering time, transgenic citrus lines ectopically expressing C. sinensis AP1 or LFY genes under control of the A. thaliana RD29A promoter exhibit precocious flowering, fruit development and viable transgenic seed formation. These transformed lines can be useful tools to reduce the time between generations to accelerate breeding.

A simple and efficient agroinfiltration method for transient gene expression in Citrus.

KEY MESSAGE: Microwounding pre-treatment facilitates agroinfiltration and transient gene expression in hard-to-agroinfiltrate citrus varieties. Agrobacterium infiltration is a widely used method for transient expression studies in plants, but this method is not used extensively in citrus because of its low efficiency. In this study, we developed an easy, cheap, and reliable agroinfiltration method for transient gene expression in citrus. A microneedle roller was used to create microscopic wounds in the leaf epidermis to facilitate agroinfiltration. Several optimization parameters were explored in this study, including the density of wounds per cm2 of abaxial leaf area, the leaf maturity grade, the effect of the Agrobacterium strain, and the length of the incubation period. Increasing the density of wounds on the leaf surface had a positive effect on transient expression. Higher transient expression levels were observed in well-expanded young leaves in comparison with older leaves. The Agrobacterium strain GV2260 was the most suitable to express a large amount of recombinant protein, and an eight- to ten-day incubation period resulted in the highest expression. Endoplasmic reticulum and cytoskeleton-targeted GFP were both successfully localized, confirming that this protocol can be used for protein subcellular localization in citrus. Finally, up to 100 ng of GFP per milligram of agroinfiltrated leaf tissue was estimated to be expressed using this method. This protocol was tested for GFP expression in five different citrus varieties with no significant statistical differences among them. This simple and easy method can speed up functional genomic studies in citrus and may be applied to other recalcitrant species with extensive epidermal cuticular wax.

The tobamovirus Turnip Vein Clearing Virus 30-kilodalton movement protein localizes to novel nuclear filaments to enhance virus infection.

Plant viruses overcome the barrier of the plant cell wall by encoding cell-to-cell movement proteins (MPs), which direct newly replicated viral genomes to, and across, the wall. The paradigm for how a single MP regulates and coordinates these activities is the Tobacco mosaic virus (TMV) 30-kDa protein (MP(TMV)). Detailed studies demonstrate that TMV multiplies exclusively in the cytoplasm and have documented associations of MP(TMV) with endoplasmic reticulum (ER) membrane, microtubules, and plasmodesmata throughout the course of infection. As TMV poorly infects Arabidopsis thaliana, Turnip vein clearing virus (TVCV) is the tobamovirus of choice for studies in this model plant. A key problem, which has contributed to confusion in the field, is the unproven assumption that the TVCV and TMV life cycles are identical. We engineered an infectious TVCV replicon that expressed a functional fluorescence-tagged MP(TVCV) and report here the unexpected discovery that MP(TVCV), beyond localizing to ER membrane and plasmodesmata, targeted to the nucleus in a nuclear localization signal (NLS)-dependent manner, where it localized to novel F-actin-containing filaments that associated with chromatin. The MP(TVCV) NLS appeared to be conserved in the subgroup 3 tobamoviruses, and our mutational analyses showed that nuclear localization of MP(TVCV) was necessary for efficient TVCV cell-to-cell movement and systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. Our studies identify a novel nuclear stage in TVCV infection and suggest that nuclear MP encoded by TVCV and other subgroup 3 tobamoviruses interacts with F-actin and chromatin to modulate host defenses or cellular physiology to favor virus movement and infection.

Stem-pitting caused by Citrus tristeza virus is associated with increased phloem occlusion.

Stem-pitting (SP) disease results from disruption of normal phloem and xylem development. In citrus, a characteristic manifestation of SP caused by Citrus tristeza virus (CTV) is phloem regeneration. We hypothesized that phloem regeneration occurs due to reduced functionality of CTV infected phloem cells. To examine phloem cell occlusions in CTV-SP, we analyzed callose and phloem-protein (PP) accumulation in Citrus macrophylla trees infected with CTV mutants exhibiting different SP phenotypes from very mild (CTVΔp13) to severe (CTVΔp33), in addition to full-length CTV and healthy plants. CTV infection was accompanied by callose and PP accumulation in the phloem. With the increase in the SP symptoms from very mild to severe, there was a constant increase in the levels of callose and PP, accompanied by an increase in PHLOEM-PROTEIN 2 and a decrease in BETA-1,3-GLUCANASE gene expression levels. These results indicate that SP symptom development is associated with increased phloem occlusion.

Subcellular dynamics and role of Arabidopsis ß-1,3-glucanases in cell-to-cell movement of tobamoviruses.

ß-1,3-Glucanases (BG) have been implicated in enhancing virus spread by degrading callose at plasmodesmata (Pd). Here, we investigate the role of Arabidopsis BG in tobamovirus spread. During Turnip vein clearing virus infection, the transcription of two pathogenesis-related (PR)-BG AtBG2 and AtBG3 increased but that of Pd-associated BG AtBG_pap did not change. In transgenic plants, AtBG2 was retained in the endoplasmic reticulum (ER) network and was not secreted. As a stress response mediated by salicylic acid, AtBG2 was secreted and appeared as a free extracellular protein localized in the entire apoplast but did not accumulate at Pd sites. At the leading edge of Tobacco mosaic virus spread, AtBG2 co-localized with the viral movement protein in the ER-derived bodies, similarly to other ER proteins, but was not secreted to the cell wall. In atbg2 mutants, callose levels at Pd and virus spread were unaffected. Likewise, AtBG2 overexpression had no effect on virus spread. However, in atbg_pap mutants, callose at Pd was increased and virus spread was reduced. Our results demonstrate that the constitutive Pd-associated BG but not the stress-regulated extracellular PR-BG are directly involved in regulation of callose at Pd and cell-to-cell transport in Arabidopsis, including the spread of viruses.

Identifying the Gut Virome of Diaphorina citri from Florida Groves.

Asian citrus psyllid (Diaphorina citri) transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the putative causative agent of citrus Huanglongbing disease (HLB). Insect-specific viruses can act against insects as their natural enemies, and recently, several D. citri-associated viruses were discovered. The insect gut plays an important role as not only a pool for diverse microbes but also as a physical barrier to prevent the spread of pathogens such as CLas. However, there is little evidence of the presence of D. citri-associated viruses in the gut and of the interaction between them and CLas. Here, we dissected psyllid guts collected from five growing regions in Florida, and the gut virome was analyzed by high throughput sequencing. Four insect viruses, including D. citri-associated C virus (DcACV), D. citri densovirus (DcDV), D. citri reovirus (DcRV), and D. citri flavi-like virus (DcFLV), were identified, and their presence in the gut, including an additional D. citri cimodo-like virus (DcCLV), were confirmed with PCR-based assays. Microscopic analysis showed that DcFLV infection leads to morphological abnormalities in the nuclear structure in the infected psyllid gut cells. The complex and diverse composition of microbiota in the psyllid gut suggests a possible interaction and dynamics between CLas and the D. citri-associated viruses. Our study identified various D. citri-associated viruses that localized in the psyllid gut and provided more information that helps to evaluate the potential vectors for manipulating CLas in the psyllid gut.

Canopy Density, but Not Bacterial Titers, Predicts Fruit Yield in Huanglongbing-Affected Sweet Orange Trees.

In Florida, almost all citrus trees are affected with Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas). We characterized various parameters of HLB-affected sweet orange trees in response to yield-improving nutritional treatment, including canopy volume, canopy density and CLas Ct values, and found that the treatment improved yield and maintained canopy density for over three years, whereas untreated HLB-affected trees declined in canopy density. The nutritional treatment did not affect CLas titer or the tree canopy volume suggesting that canopy density is a better indicator of fruit yield. To further validate the importance of canopy density, we evaluated three independent orchards (different in tree age or variety) to identify the specific traits that are correlated with fruit yields. We found that canopy density and fruit detachment force (FDF), were positively correlated with fruit yields in independent trials. Canopy density accurately distinguished between mild and severe trees in three field trials. High and low producing HLB trees had the same Ct values. Ct values did not always agree with CLas number in the phloem, as visualized by transmission electron microscopy. Our work identifies canopy density as an efficient trait to predict yields of HLB-affected trees and suggests canopy health is more relevant for yields than the CLas population.

Poor shoot and leaf growth in Huanglongbing-affected sweet orange is associated with increased investment in defenses.

Citrus disease Huanglongbing (HLB) causes sparse (thinner) canopies due to reduced leaf and shoot biomass. Herein, we present results demonstrating the possible mechanisms behind compromised leaf growth of HLB-affected 'Valencia' sweet orange trees by comparing morphological, transcriptome, and phytohormone profiles at different leaf development phases (1. buds at the start of the experiment; 2. buds on day 5; . 3. leaf emergence; 4. leaf expansion; and 5. leaf maturation) to healthy trees. Over a period of 3 months (in greenhouse conditions), HLB-affected trees had ≈40% reduction in growth traits such as tree height, number of shoots per tree, shoot length, internode length, and leaf size compared to healthy trees. In addition, buds from HLB-affected trees lagged by ≈1 week in sprouting as well as leaf growth. Throughout the leaf development, high accumulation of defense hormones, salicylic acid (SA) and abscisic acid (ABA), and low levels of growth-promoting hormone (auxin) were found in HLB-affected trees compared to healthy trees. Concomitantly, HLB-affected trees had upregulated differentially expressed genes (DEGs) encoding SA, ABA, and ethylene-related proteins in comparison to healthy trees. The total number of cells per leaf was lower in HLB-affected trees compared to healthy trees, which suggests that reduced cell division may coincide with low levels of growth-promoting hormones leading to small leaf size. Both bud dieback and leaf drop were higher in HLB-affected trees than in healthy trees, with concomitant upregulated DEGs encoding senescence-related proteins in HLB-affected trees that possibly resulted in accelerated aging and cell death. Taken together, it can be concluded that HLB-affected trees had a higher tradeoff of resources on defense over growth, leading to sparse canopies and a high tree mortality rate with HLB progression.

Comparing Machine Learning and Binary Thresholding Methods for Quantification of Callose Deposits in the Citrus Phloem.

Callose is a polysaccharide that can be fluorescently stained to study many developmental and immune functions in plants. High-throughput methods to accurately gather quantitative measurements of callose from confocal images are useful for many applications in plant biology. Previous callose quantification methods relied upon binary local thresholding, which had the disadvantage of not being able to differentiate callose in conditions with low contrast from background material. Here, a measurement approach that utilizes the Ilastik supervised machine learning imagery data collection software is described. The Ilastik software method provided superior efficiency for acquiring counts of callose deposits. We also determined the accuracy of these methods as compared to manual counts. We demonstrate that the automated software methods are both good predictors of manual counts, but that the Ilastik counts are significantly closer. Researchers can use this information to guide their choice of method to quantify callose in their work.

Survey and detection for citrus tristeza virus in Florida groves with an unconventional tool: The Asian citrus psyllid.

The citrus industry of Florida faces insurmountable challenges against the destructive diseases citrus tristeza and Huanglongbing (HLB, or citrus greening). Though the tristeza causal agent, citrus tristeza virus (CTV), has been in Florida decades longer than HLB, growers have concentrated most of their efforts on combating the more detrimental HLB. The Asian citrus psyllid (Diaphorina citri; ACP) is the insect vector of the bacterial pathogen Candidatus Liberibacter asiaticus and transmits the incurable HLB to all commercial citrus. During our searches for biological and viral controls against the ACP, we consistently detected sequences of CTV in Florida field populations of ACP. This unexpected finding led us to investigate whether ACPs collected from young shoots could be used as a tool to survey CTV in Florida citrus groves. We first surveyed for the most common CTV strains in Florida (T30, T36, and VT/T68) in citrus trees on mostly sour orange (Citrus aurantium) rootstock, the rootstock susceptible to CTV decline. Out of 968 trees sampled across five years (2018-2022), approximately 8.2% were positive for CTV, with more than half of the CTV-positive trees infected with strain T30. Simultaneously, we looked at CTV strains in ACPs during this time and found that approximately 88% of pooled adult and nymph ACPs also had CTV, with over half the positive samples having the T36 strain. As a result of the much higher CTV incidences in the ACPs, we conducted a second investigation into whether we could more easily detect the same CTV strains in ACP nymphs as in CTV-infected citrus tissue. After individually sampling 43 trees and pooling the nymphs from each tree, we detected CTV at about the same incidence in the citrus tissue and the nymphs, but with much less ACP tissue, time, and resources required for detection compared to citrus tissue. Results from this study illustrate the sustained threat of CTV to Florida citrus and demonstrate the ACP as a potential bioindicator for CTV.

Diaphorina citri flavi-like virus localization, transmission, and association with Candidatus Liberibacter asiaticus in its psyllid host.

Huanglongbing is caused by Candidatus Liberibacter asiaticus (CLas) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with CLas are unknown. DcFLV was found in various organs of D. citri, including the midgut and salivary glands, where it co-localized with CLas. CLas-infected nymphs had the highest DcFLV titers compared to the infected adults and CLas-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and CLas were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with CLas in the vector. These results provide new insights on the interactions between DcFLV, the D. citri, and CLas.

Comparative transcriptome analysis of Citrus macrophylla tree infected with Citrus tristeza virus stem pitting mutants provides new insight into the role of phloem regeneration in stem pitting disease.

Stem pitting is a complex and economically important virus-associated disease of perennial woody plants. Molecular mechanisms and pathways occurring during virus-plant interaction that result in this phenomenon are still obscure. Previous studies indicated that different Citrus tristeza virus (CTV) mutants induce defined stem pitting phenotypes ranging from mild (CTVΔp13) to severe (CTVΔp33) in Citrus macrophylla trees. In this study, we conducted comparative transcriptome analyses of C. macrophylla trees infected with CTV mutants (CTVΔp13 and CTVΔp33) and a full-length virus in comparison to healthy plants as control. The mild CTV stem pitting mutant had very few differentially expressed genes (DEGs) related to plant defense mechanism and plant growth and development. In contrast, substantial gene expression changes were observed in plants infected with the severe mutant and the full-length virus, indicating that both the p13 and p33 proteins of CTV acted as a regulator of symptom production by activating and modulating plant responses, respectively. The analysis of transcriptome data for CTVΔp33 and the full-length virus suggested that xylem specification has been blocked by detecting several genes encoding xylem, cell wall and lignin degradation, and cell wall loosening enzymes. Furthermore, stem pitting was accompanied by downregulation of transcription factors involved in regulation of xylem differentiation and downregulation of some genes involved in lignin biosynthesis, showing that the xylem differentiation and specification program has been shut off. Upregulation of genes encoding transcription factors associated with phloem and cambium development indicated the activation of this program in stem pitting disease. Furthermore, we detected the induction of several DEGs encoding proteins associated with cell cycle re-entry such as chromatin remodeling factors and cyclin, and histone modification. This kind of expression pattern of genes related to xylem differentiation and specification, phloem and cambium development, and cell cycle re-entry is demonstrated during secondary vascular tissue (SVT) regeneration. The microscopy analysis confirmed that the regeneration of new phloem is associated with stem pitting phenotypes. The findings of this study, thus, provide evidence for the association between stem pitting phenotypes and SVT regeneration, suggesting that the expression of these genes might play important roles in development of stem pitting symptoms. Overall, our findings suggest that phloem regeneration contributes to development of stem pitting symptoms.