A chromosome-level phased genome enabling allele-level studies in sweet orange: a case study on citrus Huanglongbing tolerance.

Sweet orange originated from the introgressive hybridizations of pummelo and mandarin resulting in a highly heterozygous genome. How alleles from the two species cooperate in shaping sweet orange phenotypes under distinct circumstances is unknown. Here, we assembled a chromosome-level phased diploid Valencia sweet orange (DVS) genome with over 99.999% base accuracy and 99.2% gene annotation BUSCO completeness. DVS enables allele-level studies for sweet orange and other hybrids between pummelo and mandarin. We first configured an allele-aware transcriptomic profiling pipeline and applied it to 740 sweet orange transcriptomes. On average, 32.5% of genes have a significantly biased allelic expression in the transcriptomes. Different cultivars, transgenic lineages, tissues, development stages, and disease status all impacted allelic expressions and resulted in diversified allelic expression patterns in sweet orange, but particularly citrus Huanglongbing (HLB) shifted the allelic expression of hundreds of genes in leaves and calyx abscission zones. In addition, we detected allelic structural mutations in an HLB-tolerant mutant (T19) and a more sensitive mutant (T78) through long-read sequencing. The irradiation-induced structural mutations mostly involved double-strand breaks, while most spontaneous structural mutations were transposon insertions. In the mutants, most genes with significant allelic expression ratio alterations (≥1.5-fold) were directly affected by those structural mutations. In T19, alleles located at a translocated segment terminal were upregulated, including CsDnaJ, CsHSP17.4B, and CsCEBPZ. Their upregulation is inferred to keep phloem protein homeostasis under the stress from HLB and enable subsequent stress responses observed in T19. DVS will advance allelic level studies in citrus.

Identification of a Chromosomal Deletion Mutation and the Dynamics of Two Major Populations of 'Candidatus Liberibacter asiaticus' in Its Hosts.

'Candidatus Liberibacter asiaticus' (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.

Selection and Evaluation of a Thornless and HLB-Tolerant Bud-Sport of Pummelo Citrus With an Emphasis on Molecular Mechanisms.

The selection of elite bud-sports is an important breeding approach in horticulture. We discovered and evaluated a thornless pummelo bud-sport (TL) that grew more vigorously and was more tolerant to Huanglongbing (HLB) than the thorny wild type (W). To reveal the underlying molecular mechanisms, we carried out whole-genome sequencing of W, and transcriptome comparisons of W, TL, and partially recovered thorny "mutants" (T). The results showed W, TL, and T varied in gene expression, allelic expression, and alternative splicing. Most genes/pathways with significantly altered expression in TL compared to W remained similarly altered in T. Pathway and gene ontology enrichment analysis revealed that the expression of multiple pathways, including photosynthesis and cell wall biosynthesis, was altered among the three genotypes. Remarkably, two polar auxin transporter genes, PIN7 and LAX3, were expressed at a significantly lower level in TL than in both W and T, implying alternation of polar auxin transport in TL may be responsible for the vigorous growth and thornless phenotype. Furthermore, 131 and 68 plant defense-related genes were significantly upregulated and downregulated, respectively, in TL and T compared with W. These genes may be involved in enhanced salicylic acid (SA) dependent defense and repression of defense inducing callose deposition and programmed cell death. Overall, these results indicated that the phenotype changes of the TL bud-sport were associated with tremendous transcriptome alterations, providing new clues and targets for breeding and gene editing for citrus improvement.

Highly Sensitive and Rapid Detection of Citrus Huanglongbing Pathogen ('Candidatus Liberibacter asiaticus') Using Cas12a-Based Methods.

Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, 'Candidatus Liberibacter asiaticus' (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid-based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.

Field Evaluation of Chemotherapy on HLB-Affected Citrus Trees With Emphasis on Fruit Yield and Quality.

Huanglongbing (HLB) is one of the most devastating diseases of citrus, which is associated with Candidatus Liberibacter asiaticus (Las) in the United States. To date, no effective antimicrobial compound is commercially available to control the disease. In this study, we investigated the effects of different antimicrobial chemicals with suitable surfactants on HLB-affected matured citrus trees with emphasis on the fruit yield and quality. Each treatment was applied three times in a 2-week interval during the spring flush period, one time in summer and three times during the autumn flushing period. We extensively examined different parameters such as pathogenic index, disease index, tree canopy, fruit yield, quality, and nutritional status. The results showed that among the treatments, penicillin (PEN) with surfactant was most effective in suppressing Las titer in infected citrus trees, followed by Fosetyl-Al (ALI), Carvacrol (CARV), and Validamycin (VA). Fruit quality analysis revealed that PEN treatment increased the soluble solids content (SSC), whereas Oxytetracycline (OXY) treatment significantly reduced titratable acidity (TA) level and increased the SSC/TA ratio compared to the control. Nutrient analysis showed increased N and Zn levels in ALI and PEN treatments, and OXY treatment increased leaf P, K, S, and Mg levels compared to untreated control. Furthermore, B, Ca, Cu, Fe, and Mn in leaves were reduced in all chemical treatments than that of the untreated control. These findings revealed that some of the chemical treatments were able to suppress Las pathogen, enhance nutritional status in leaves, and improve tree growth and fruit quality of HLB-affected trees.

Metagenomic Analysis Reveals the Mechanism for the Observed Increase in Antibacterial Activity of Penicillin against Uncultured Bacteria Candidatus Liberibacter asiaticus Relative to Oxytetracycline in Planta.

Citrus huanglongbing (HLB) is a devastating disease for the citrus industry. The previous studies demonstrated that oxytetracycline and penicillin are effective antibiotics against Candidatus Liberibacter asiaticus (CLas). However, since CLas is uncultured, the mechanisms of action of antibiotics against CLas are still unclear. It was recently reported that the endophytic microbial communities are associated with the progression of citrus HLB after oxytetracycline and penicillin treatment. Therefore, we hypothesize that penicillin has greater antibacterial activity against CLas than oxytetracycline, which may be associated with the alteration of the structure and function of endophytic microbial communities in HLB-affected citrus in response to these antibiotics. To test this hypothesis, the microbiome of HLB-affected citrus leaves treated with these two antibiotics was analyzed using a metagenomic method. Our results indicate that the microbial structure and function in HLB-affected citrus were altered by these two antibiotics. The relative abundance of beneficial bacterial species, including Streptomyces avermitilis and Bradyrhizobium, was higher in penicillin-treated plants compared to those treated with oxytetracycline, and the relative abundance of the bacterial species (such as Propionibacterium acnes and Synechocystis sp PCC 6803) associated with CLas survival was lower for penicillin-treated plants compared to oxytetracycline-treated plants. These results indicate that penicillin has greater antibacterial activity against CLas. Based on the metagenomic analysis, this study elucidated the mechanism for the observed increase in antibacterial activity of penicillin against CLas. The data presented here are not only invaluable for developing eco-friendly and effective biocontrol strategies to combat citrus HLB, but also provide a method for revealing mechanism of antimicrobial against uncultured bacteria in host.

'Candidatus Liberibacter Asiaticus' SDE1 Effector Induces Huanglongbing Chlorosis by Downregulating Host DDX3 Gene.

'Candidatus Liberibacter asiaticus' (CLas) is the pathogenic bacterium that causes the disease Huanglongbing (HLB) in citrus and some model plants, such as Nicotiana benthamiana. After infection, CLas releases a set of effectors to modulate host responses. One of these critical effectors is Sec-delivered effector 1 (SDE1), which induces chlorosis and cell death in N. benthamiana. In this study, we revealed the DEAD-box RNA helicase (DDX3) interacts with SDE1. Gene silencing study revealed that knockdown of the NbDDX3 gene triggers leaf chlorosis, mimicking the primary symptom of CLas infection in N. benthamiana. The interactions between SDE1 and NbDDX3 were localized in the cell membrane. Overexpression of SDE1 resulted in suppression of NbDDX3 gene expression in N. benthamiana, which suggests a critical role of SDE1 in modulating NbDDX3 expression. Furthermore, we verified the interaction of SDE1 with citrus DDX3 (CsDDX3), and demonstrated that the expression of the CsDDX3 gene was significantly reduced in HLB-affected yellowing and mottled leaves of citrus. Thus, we provide molecular evidence that the downregulation of the host DDX3 gene is a crucial mechanism of leaf chlorosis in HLB-affected plants. The identification of CsDDX3 as a critical target of SDE1 and its association with HLB symptom development indicates that the DDX3 gene is an important target for gene editing, to interrupt the interaction between DDX3 and SDE1, and therefore interfere host susceptibility.

Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and integration with disease control.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ∼2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

Antimicrobial Compounds Effective against Candidatus Liberibacter asiaticus Discovered via Graft-based Assay in Citrus.

Huanglongbing (HLB), the most destructive citrus disease, is caused by three species of phloem-limited Candidatus Liberibacter. Chemical control is a critical short-term strategy against Candidatus Liberibacter asiaticus (Las). Currently, application of antibiotics in agricultural practices is limited due to public concerns regarding emergence of antibiotic-resistant bacteria and potential side effects in humans. The present study screened 39 antimicrobials (non-antibiotics) for effectiveness against Las using an optimized graft-based screening system. Results of principal component, hierarchical clustering and membership function analyses demonstrated that 39 antimicrobials were clustered into three groups: "effective" (Group I), "partly effective" (Group II), and "ineffective" (Group III). Despite different modes of action, 8 antimicrobials (aluminum hydroxide, D,L-buthionine sulfoximine, nicotine, surfactin from Bacillus subtilis, SilverDYNE, colloidal silver, EBI-601, and EBI-602), were all as highly effective at eliminating or suppressing Las, showing both the lowest Las infection rates and titers in treated scions and inoculated rootstock. The ineffective group, which included 21 antimicrobials, did not eliminate or suppress Las, resulting in plants with increased titers of Candidatus Liberibacter. The other 10 antimicrobials partly eliminated/suppressed Las in treated and graft-inoculated plants. These effective antimicrobials are potential candidates for HLB control either via rescuing infected citrus germplasms or restricted field application.

Molecular mechanisms underlying heat or tetracycline treatments for citrus HLB control.

Huanglongbing (HLB), a destructive plant bacterial disease, severely impedes worldwide citrus production. In our previous reports, we revealed the molecular mechanisms of host plant responses that underlie thermotherapy against HLB. In this study, we investigated the molecular mechanism underlying heat or tetracycline treatments on the HLB bacterium, 'Candidatus Liberibacter asiaticus' (Las) by focusing on Las prophage/phage conversion under stress conditions. By comparing the prophage FP1 and FP2 copy number to the copy number of 16S rDNA in HLB-affected plants, we found that the relative copy number of both FP1 and FP2 increased significantly, ranging from 3.4- to 6.7-fold change when Las-infected samples underwent a temperature shift from 23 to 37, 42 or 45 °C. When treated with tetracycline at 50-150 and 200-250 µg/ml, respectively, the relative copy number of both FP1 and FP2 increased by 3.4- to 6.0-fold. In addition, analyses of Las prophage structural gene and antirepressor gene copy numbers showed similar trends for all treatments. Furthermore, transmission electron microscopy provided direct evidence of lysogenic to lytic conversion upon temperature increase. These results not only provide new insight into the molecular mechanisms underlying heat or tetracycline treatment but also suggest a novel HLB control strategy by enhancing the endogenous conversion from Las prophages to phages.

LasΔ5315 Effector Induces Extreme Starch Accumulation and Chlorosis as Ca. Liberibacter asiaticus Infection in Nicotiana benthamiana.

Huanglongbing (HLB), a destructive plant bacterial disease, severely impedes worldwide citrus production. HLB is associated with a phloem-limited α-proteobacterium, Candidatus Liberibacter asiaticus (Las). Las infection causes yellow shoots and blotchy mottle on leaves and is associated with excessive starch accumulation. However, the mechanisms underlying the starch accumulation remain unknown. We previously showed that the Las5315mp effector induced callose deposition and cell death in Nicotiana benthamiana. In this study, we demonstrated that Las can experimentally infect N. benthamiana via dodder transmission. Furthermore, we revealed another key function of the Las5315 effector by demonstrating that transient expression of the truncated form of the effector, LasΔ5315, induced excessive starch accumulation by 6 fold after 8 dpi in N. benthamiana after removal of the chloroplast transit peptide from the Las5315mp. The induction mechanisms of LasΔ5315 in N. benthamiana were attributed to the up-regulation of ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, and starch branching enzyme for increasing starch production, and to the significant down-regulation of the starch degradation enzymes: alpha-glucosidase, alpha-amylase, and glycosyl hydrolase for decreasing starch degradation. This is the first report that Las can infect the model plant N. benthamiana. Using this model plant, we demonstrated that the LasΔ5315 effector caused the most prominent HLB symptoms, starch accumulation and chlorosis as Las infection in N. benthamiana. Altogether the Las 5315 effector is critical for Las pathogenesis, and therefore, an important target for interference.

Field Evaluation of Integrated Management for Mitigating Citrus Huanglongbing in Florida.

Citrus huanglongbing (HLB) is extremely difficult to control because the psyllid-transmitted bacterial pathogen resides inside the citrus phloem and the disease is systemic. In Florida, the nine billion dollar citrus industry has been significantly impacted by severe HLB epidemics. To combat citrus HLB, in this study we implemented an integrated strategy that includes chemotherapy, thermotherapy, and additional nutrition treatment in three different field trials over three consecutive years. In these trials, only trees already showing HLB symptoms with Ct values ranging from 25.1 to 27.7 were selected for treatments. To assess the complex interactions, we used several methods for evaluating the effectiveness of integrated management, including the slopes (b) of the Ct increase (dy/dt), the pathogenic index (PI) and the decline index (DI) from Ct value and tree scores, and the therapeutic efficacies from PI and DI. This comprehensive analysis showed that most of the tested chemicals were effective to some degree in killing or suppressing the Las bacterium, with higher therapeutic efficacies seen for Grove B, where citrus trees were severely affected by HLB, and it had a higher number of psyllids, relative to Grove E and P in the first 2 years. Trunk-injected penicillin G potassium was the most effective chemical treatment in all groves, followed by Oxytetracycline Calcium Complex, and Silver Nitrate delivered as foliar sprays. Although the steam heat treatment and additional nutrition did not eliminate or suppress Las over the long term, these treatments did positively affect tree growth and recovery in the short term. Overall, our results provide new insights into HLB control method and strategy for integrated management for HLB epidemic plantations.

Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliata and its hybrids.

Huanglongbing (HLB) is the most destructive bacterial disease of citrus worldwide. While most citrus varieties are susceptible to HLB, Poncirus trifoliata, a close relative of Citrus, and some of its hybrids with Citrus are tolerant to HLB. No specific HLB tolerance genes have been identified in P. trifoliata but recent studies have shown that constitutive disease resistance (CDR) genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus (CLas), the pathogen of HLB. The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus. We identified 17 CDR genes in two citrus genomes, deduced their structures, and investigated their phylogenetic relationships. We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events. Through genome resequencing and transcriptome sequencing, we identified eight CDR genes in the Poncirus genome (PtCDR1-PtCDR8). The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7. Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes. PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant, Poncirus hybrids as revealed by real-time PCR analysis. These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus and enhances canopy growth by altering gene expression profiles in HLB-affected citrus plants.

Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with 'Candidatus Liberibacter asiaticus' (Las) in the United States. Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42 °C for a minimum of 48 h. To determine the feasibility and effectiveness of solar thermotherapy in the field, portable plastic enclosures were placed over commercial and residential citrus, exposing trees to high temperatures through solarization. Within 3-6 weeks after treatment, most trees responded with vigorous new growth. Las titer in new growth was greatly reduced for 18-36 months after treatment. Unlike with potted trees, exposure to high heat did not eradicate the Las population under field conditions. This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers, and the failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB.

Molecular mechanisms behind the accumulation of ATP and H2O2 in citrus plants in response to 'Candidatus Liberibacter asiaticus' infection.

Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells, such as mitochondria and chloroplasts to manipulate host immunity and physiology. These organelles are responsible for the synthesis of adenosine triphosphate (ATP) and have a critical role in plant immune signaling during hydrogen peroxide (H2O2) production. In this study, we investigated H2O2 and ATP accumulation in relation to citrus huanglongbing (HLB) in addition to revealing the expression profiles of genes critical for the production and detoxification of H2O2 and ATP synthesis. We also found that as ATP and H2O2 concentrations increased in the leaf, so did the severity of the HLB symptoms, a trend that remained consistent among the four different citrus varieties tested. Furthermore, the upregulation of ATP synthase, a key enzyme for energy conversion, may contribute to the accumulation of ATP in infected tissues, whereas downregulation of the H2O2 detoxification system may cause oxidative damage to plant macromolecules and cell structures. This may explain the cause of some of the HLB symptoms such as chlorosis or leaf discoloration. The findings in this study highlight important molecular and physiological mechanisms involved in the host plants' response to Las infection and provide new targets for interrupting the disease cycle.

Effects of thermal processing and pulp filtration on physical, chemical and sensory properties of winter melon juice.

BACKGROUND: Winter melon (Benincasa hispida) is a widely consumed crop in Asia, and believed to impart special benefits to human health. The nutritional composition and sensory properties of four juice types, resulting from a combination of pulp levels (low/high pulp, LP/HP) and thermal processing (with/without boiling, B/NB), LPNB, HPNB, LPB and HPB, were compared. RESULTS: The juices had low sugars (< 20 g kg-1 ) and low titratable acidity (about 2 g kg-1 ). The insoluble solids, glucose, fructose and citric acid content in LP juice were significantly lower than in HP juice. The phenolic amino acids phenylalanine, tyrosine, and tryptophan were detected at 10-45 mg L-1 levels, and the antioxidant activity ranged from 36 to 49 mg gallic acid L-1 . C6 and C9 aldehydes were mainly found in HP juice, and boiling induced the accumulation of sulfur compounds and C5 aldehydes. The LPNB juice showed the highest acceptability in the sensory panel. The frozen concentrated orange juice (FCOJ) reconstituted with LPNB was preferable to regular FCOJ for 31% of panelists and not different for 20% of panelists. CONCLUSION: The low sugar/low acid LPNB juice with 'fresh' flavor could be developed to replace water for reconstituting FCOJ with enhanced nutritional value. © 2016 Society of Chemical Industry.

Proteomics analysis reveals novel host molecular mechanisms associated with thermotherapy of 'Ca. Liberibacter asiaticus'-infected citrus plants.

BACKGROUND: Citrus Huanglongbing (HLB), which is linked to the bacterial pathogen 'Ca. Liberibacter asiaticus' (Las), is the most devastating disease of citrus plants, and longer-term control measures via breeding or genetic engineering have been unwieldy because all cultivated citrus species are susceptible to the disease. However, the degree of susceptibility varies among citrus species, which has prompted efforts to identify potential Las resistance/tolerance-related genes in citrus plants for application in breeding or genetic engineering programs. Plant exposure to one form of stress has been shown to serendipitously induce innate resistance to other forms of stress and a recent study showed that continuous heat treatment (40 to 42 °C) reduced Las titer and HLB-associated symptoms in citrus seedlings. The goal of the present study was to apply comparative proteomics analysis via 2-DE and mass spectrometry to elucidate the molecular processes associated with heat-induced mitigation of HLB in citrus plants. Healthy or Las-infected citrus grapefruit plants were exposed to room temperature or to continuous heat treatment of 40 °C for 6 days. RESULTS: An exhaustive total protein extraction process facilitated the identification of 107 differentially-expressed proteins in response to Las and/or heat treatment, which included a strong up-regulation of chaperones including small (23.6, 18.5 and 17.9 kDa) heat shock proteins, a HSP70-like protein and a ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO)-binding 60 kDa chaperonin, particularly in response to heat treatment. Other proteins that were generally down-regulated due to Las infection but up-regulated in response to heat treatment include RuBisCO activase, chlorophyll a/b binding protein, glucosidase II beta subunit-like protein, a putative lipoxygenase protein, a ferritin-like protein, and a glutathione S-transferase. CONCLUSIONS: The differentially-expressed proteins identified in this study highlights a premier characterization of the molecular mechanisms potentially involved in the reversal of Las-induced pathogenicity processes in citrus plants and are hence proposed targets for application towards the development of cisgenic Las-resistant/tolerant citrus plants.

Transcriptome Profiling of Huanglongbing (HLB) Tolerant and Susceptible Citrus Plants Reveals the Role of Basal Resistance in HLB Tolerance.

Huanglongbing (HLB) is currently the most destructive disease of citrus worldwide. Although there is no immune cultivar, field tolerance to HLB within citrus and citrus relatives has been observed at the USDA Picos farm at Ft. Pierce, Florida, where plants have been exposed to a very high level of HLB pressure since 2006. In this study, we used RNA-Seq to evaluate expression differences between two closely related cultivars after HLB infection: HLB-tolerant "Jackson" grapefruit-like-hybrid trees and HLB susceptible "Marsh" grapefruit trees. A total of 686 genes were differentially expressed (DE) between the two cultivars. Among them, 247 genes were up-expressed and 439 were down-expressed in tolerant citrus trees. We also identified a total of 619 genes with significant differential expression of alternative splicing isoforms between HLB tolerant and HLB susceptible citrus trees. We analyzed the functional categories of DE genes using two methods, and revealed that multiple pathways have been suppressed or activated in the HLB tolerant citrus trees, which lead to the activation of the basal resistance or immunity of citrus plants. We have experimentally verified the expressions of 14 up-expressed genes and 19 down-expressed genes on HLB-tolerant "Jackson" trees and HLB-susceptible "Marsh" trees using real time PCR. The results showed that the expression of most genes were in agreement with the RNA-Seq results. This study provided new insights into HLB-tolerance and useful guidance for breeding HLB-tolerant citrus in the future.