Uncovering Interactions between Plant Metabolism and Plant-Associated Bacteria in Huanglongbing-Affected Citrus Cultivars Using Multiomics Analysis and Machine Learning.

Huanglongbing (HLB) is a highly destructive disease that inflicts significant economic losses on the citrus industry worldwide but with no cure available. However, microbiomes formulated by citrus plants may serve as disease antagonists, increasing the level of HLB tolerance. This study established an integrated analysis of untargeted metabolomics and microbiomics data for different citrus cultivars, providing critical insights into the interactions between plant metabolism and plant-associated bacteria in the development of HLB. Machine learning models were applied to screen important metabolites and bacteria in multiple citrus materials, and the selected metabolites were then analyzed to identify essential pathways enriched in the plant and to correlate with the selected bacteria. Results demonstrated that the regulation of plant pathways, especially ABC transporters and ubiquinone and other terpene-ubiquinone biosynthesis pathways, could affect the microbial community structure, indicating potential solutions for controlling HLB by modulating bacteria in citrus plants or breeding tolerant citrus cultivars.

Plant mitochondrial introns as genetic markers - conservation and variation.

Plant genomes are comprised of nuclear, plastid and mitochondrial components characterized by different patterns of inheritance and evolution. Genetic markers from the three genomes provide complementary tools for investigations of inheritance, genetic relationships and phenotypic contributions. Plant mitochondrial genomes are challenging for universal marker development because they are highly variable in terms of size, gene order and intergenic sequences and highly conserved with respect to protein-coding sequences. PCR amplification of introns with primers that anneal to conserved, flanking exons is effective for the development of polymorphic nuclear genome markers. The potential for plant mitochondrial intron polymorphisms to distinguish between congeneric species or intraspecific varieties has not been systematically investigated and is possibly constrained by requirements for intron secondary structure and interactions with co-evolved organelle intron splicing factors. To explore the potential for broadly applicable plant mitochondrial intron markers, PCR primer sets based upon conserved sequences flanking 11 introns common to seven angiosperm species were tested across a range of plant orders. PCR-amplified introns were screened for indel polymorphisms among a group of cross-compatible Citrus species and relatives; two Raphanus sativus mitotypes; representatives of the two Phaseolus vulgaris gene pools; and congeneric pairs of Cynodon, Cenchrus, Solanum, and Vaccinium species. All introns were successfully amplified from each plant entry. Length polymorphisms distinguishable by gel electrophoresis were common among genera but infrequent within genera. Sequencing of three introns amplified from 16 entries identified additional short indel polymorphisms and nucleotide substitutions that separated Citrus, Cynodon, Cenchrus and Vaccinium congeners, but failed to distinguish Solanum congeners or representatives of the Phaseolus vulgaris major gene pools. The ability of primer sets to amplify a wider range of plant species' introns and the presence of intron polymorphisms that distinguish congeners was confirmed by in silico analysis. While mitochondrial intron variation is limited in comparison to nuclear introns, these exon-based primer sets provide robust tools for the amplification of mitochondrial introns across a wide range of plant species wherein useful polymorphisms can be identified.

Role of long non-coding RNA in regulatory network response to Candidatus Liberibacter asiaticus in citrus.

Long non-coding RNAs (lncRNAs) serve as crucial regulators in plant response to various diseases, while none have been systematically identified and characterized in response to citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) bacteria. Here, we comprehensively investigated the transcriptional and regulatory dynamics of the lncRNAs in response to CLas. Samples were collected from leaf midribs of CLas- and mock-inoculated HLB-tolerant rough lemon (Citrus jambhiri) and HLB-sensitive sweet orange (C. sinensis) at week 0, 7, 17, and 34 following inoculation using CLas+ budwood of three biological replicates in the greenhouse. A total of 8,742 lncRNAs, including 2,529 novel lncRNAs, were identified from RNA-seq data with rRNA-removed from strand-specific libraries. Genomic variation analyses of conserved lncRNAs from 38 citrus accessions showed that 26 single nucleotide polymorphisms (SNPs) were significantly correlated with HLB. In addition, lncRNA-mRNA weighted gene co-expression network analysis (WGCNA) showed a significant module correlated with CLas-inoculation in rough lemon. Notably, the most significant LNC_28805 and multiple co-expressed genes related to plant defense in the module were targeted by miRNA5021, suggesting that LNC28805 might compete with endogenous miR5021 to maintain the homeostasis of immune gene expression levels. Candidate WRKY33 and SYP121 genes targeted by miRNA5021 were identified as two key hub genes interacting with bacteria pathogen response genes based on the prediction of protein-protein interaction (PPI) network. These two genes were also found within HLB-associated QTL in linkage group 6. Overall, our findings provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation.

Highly efficient hairy root genetic transformation and applications in citrus.

Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by Agrobacterium tumefaciens, is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by A. rhizogenes K599 is described. This technology can be applied to multiple citrus genotypes, and only 2-8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that A. rhizogenes-mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ACD2) for obtaining "hard-to-get" transgenic root tissue. Furthermore, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

Genome-Wide Association Study of Healthful Flavonoids among Diverse Mandarin Accessions.

Mandarins have many unique flavonoids with documented health benefits and that help to prevent chronic human diseases. Flavonoids are difficult to measure and cannot be phenotyped without the use of specialized equipment; consequently, citrus breeders have not used flavonoid contents as selection criteria to develop cultivars with increased benefits for human health or increased tolerance to diseases. In this study, peel, pulp, and seed samples collected from many mandarin accessions and their hybrids were analyzed for the presence of selected flavonoids with documented human health benefits. A genome-wide association study (GWAS) was used to identify SNPs associated with biosynthesis of flavonoids in these mandarin accessions, and there were 420 significant SNPs were found to be associated with 28 compounds in peel, pulp, or seed samples. Four candidate genes involved in flavonoid biosynthesis were identified by enrichment analysis. SNPs that were found to be associated with compounds in pulp samples have the potential to be used as markers to select mandarins with improved phytonutrient content to benefit human health. Mandarin cultivars bred with increased flavonoid content may provide value to growers and consumers.

Histone Acetyltransferases and Deacetylases Are Required for Virulence, Conidiation, DNA Damage Repair, and Multiple Stresses Resistance of Alternaria alternata.

Histone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) (Gcn5, RTT109, Elp3, Sas3, Sas2, Nat3) and 6 histone deacetylases (HDACs) (Hos2, Rpd3, Hda1, Hos3, Hst2, Sir2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants (ΔGcn5, ΔRTT109, ΔElp3, ΔSas3, ΔNat3, ΔHos2, and ΔRpd3) displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress (Sas3, Gcn5, Elp3, RTT109, Hos2), osmotic stress (Sas3, Gcn5, RTT109, Hos2), cell wall-targeting agents (Sas3, Gcn5, Hos2), and fungicide (Gcn5, Hos2). ΔGcn5, ΔSas3, and ΔHos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in ΔGcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover, ΔRTT109, ΔGcn5, and ΔHos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in ΔGcn5 and ΔHos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata.

Comparative Leaf Volatile Profiles of Two Contrasting Mandarin Cultivars against Candidatus Liberibacter asiaticus Infection Illustrate Huanglongbing Tolerance Mechanisms.

Huanglongbing (HLB), presumably caused by Candidatus Liberibacter asiaticus (CaLas), is a devastating citrus disease worldwide. While all citrus are affected by HLB, some cultivars display greater tolerance; however, the underlying mechanisms are not fully understood. Here, volatile changes in HLB-tolerant LB8-9 Sugar Belle (SB) and HLB-sensitive Murcott mandarins after CaLas infection were comprehensively compared to determine if specific volatiles are associated with HLB responses and to discern the underlying tolerance mechanisms. These cultivars emitted qualitatively and quantitatively different volatiles in response to HLB induced by artificial graft or natural psyllid inoculation. Increasing amounts of total volatiles and de novo-synthesized new volatiles were two key responses to HLB of both cultivars. Markers potentially associated with HLB and host susceptibility were identified. Terpenoid biosynthetic pathway, green leaf volatile, and thymol metabolic pathways responsive to CaLas infection were dramatically altered. SB mandarin allows simultaneous defense and growth, contributing to its greater HLB tolerance.

The Mechanism of Citrus Host Defense Response Repression at Early Stages of Infection by Feeding of Diaphorina citri Transmitting Candidatus Liberibacter asiaticus.

Citrus Huanglongbing (HLB) is the most devastating disease of citrus, presumably caused by "Candidatus Liberibacter asiaticus" (CaLas). Although transcriptomic profiling of HLB-affected citrus plants has been studied extensively, the initial steps in pathogenesis have not been fully understood. In this study, RNA sequencing (RNA-seq) was used to compare very early transcriptional changes in the response of Valencia sweet orange (VAL) to CaLas after being fed by the vector, Diaphorina citri (Asian citrus psyllid, or ACP). The results suggest the existence of a delayed defense reaction against the infective vector in VAL, while the attack by the healthy vector prompted immediate and substantial transcriptomic changes that led to the rapid erection of active defenses. Moreover, in the presence of CaLas-infected psyllids, several downregulated differentially expressed genes (DEGs) were identified on the pathways, such as signaling, transcription factor, hormone, defense, and photosynthesis-related pathways at 1 day post-infestation (dpi). Surprisingly, a burst of DEGs (6,055) was detected at 5 dpi, including both upregulated and downregulated DEGs on the defense-related and secondary metabolic pathways, and severely downregulated DEGs on the photosynthesis-related pathways. Very interestingly, a significant number of those downregulated DEGs required ATP binding for the activation of phosphate as substrate; meanwhile, abundant highly upregulated DEGs were detected on the ATP biosynthetic and glycolytic pathways. These findings highlight the energy requirement of CaLas virulence processes. The emerging picture is that CaLas not only employs virulence strategies to subvert the host cell immunity, but the fast-replicating CaLas also actively rewires host cellular metabolic pathways to obtain the necessary energy and molecular building blocks to support virulence and the replication process. Taken together, the very early response of citrus to the CaLas, vectored by infective ACP, was evaluated for the first time, thus allowing the changes in gene expression relating to the primary mechanisms of susceptibility and host-pathogen interactions to be studied, and without the secondary effects caused by the development of complex whole plant symptoms.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Effect of fruit maturity on volatiles and sensory descriptors of four mandarin hybrids.

Mandarins (or tangerines) are mainly consumed as fresh fruits due to the ease of peeling and desirable flavor. Sweetness, acidity, and flavor of mandarin are the most important criteria for consumer preference. The objective of this study was to evaluate the effects of harvest date on sensory and chemical components of four mandarin cultivars (Murcott, 411, Temple, and 'LB8-9' Sugar Belle®). Volatiles were extracted from the headspace of juice samples with solid phase microextraction (SPME) and analyzed using gas chromatography-mass spectrometry (GC-MS). The optimum harvest window for eating quality of 411 was late January to mid-February (soluble solids content [SSC]/titratable acidity [TA]: 11.3 to 14.0), Sugar Belle® fruits were best tasted when harvested from mid- to end of January (SSC/TA: 14.1 to 16.1), and February was the best month for harvesting Murcott (SSC/TA: 13.10 to 18.0) and Temple (SSC/TA:10.3 to 12.50). Sensory perception of sweetness, ripeness, and juiciness increased as SSC/TA increased while sourness and bitterness decreased. Pumpkin flavor, an indicator of overripe fruit, was mainly noticed late in the season. Tangerine flavor tended to decrease, whereas fruity-noncitrus flavor tended to increase with fruit maturity. Monoterpenes were the most abundant volatiles and tended to decrease with fruit maturity, whereas alcohols, esters, and aldehydes increase. Aldehydes, esters, and alcohols were positively correlated with sweetness, ripeness, juiciness, and fruity characteristics, and negatively with sourness and bitterness. On the other hand, monoterpenes were positively correlated with bitterness and tangerine flavor, and negatively correlated with sweetness and fruity-noncitrus flavor. The highest number of esters was found in Temple, whereas Murcott and 411 were high in aldehydes.

Novel assembly strategy cracks open the mysteries of walnut genome evolution.

High quality chromosome-scale assemblies from an interspecific hybrid between walnut and a wild relative reveal the persistence of asymmetric fractionation between the sub-genomes and suggest a late-Miocene origin for the genus Juglans.

Phloem Regeneration Is a Mechanism for Huanglongbing-Tolerance of "Bearss" Lemon and "LB8-9" Sugar Belle® Mandarin.

Huanglongbing (HLB) is an extremely destructive and lethal disease of citrus worldwide, presumably caused by phloem-limited bacteria, Candidatus Liberibacter asiaticus (CLas). The widespread invasiveness of the HLB pathogen and lack of natural HLB-resistant citrus cultivars have underscored the need for identifying tolerant citrus genotypes to support the current citrus industry's survival and potentially to lead to future natural HLB resistance. In this study, transverse sections of leaf lamina and midribs were examined with light and epifluorescence microscopy to determine anatomical characteristics that underlie HLB-tolerant mechanisms operating among "Bearss" lemon, "LB8-9" Sugar Belle® mandarin, and its sibling trees compared with HLB-sensitive "Valencia" sweet orange. The common anatomical aberrations observed in all CLas-infected varieties are as follows: phloem necrosis, hypertrophic phloem parenchyma cells, phloem plugging with abundant callose depositions, phloem collapse with cell wall distortion and thickening, excessive starch accumulation, and sometimes even cambium degeneration. Anatomical distribution of starch accumulation even extended to tracheid elements. Although there were physical, morphological, and pathological similarities in the examined foliage, internal structural preservation in "Bearss" lemon and "LB8-9" Sugar Belle® mandarin was superior compared with HLB-sensitive "Valencia" sweet orange and siblings of "LB8-9" Sugar Belle® mandarin. Intriguingly, there was substantial phloem regeneration in the tolerant types that may compensate for the dysfunctional phloem, in comparison with the sensitive selections. The lower levels of phloem disruption, together with greater phloem regeneration, are two key elements that contribute to HLB tolerance in diverse citrus cultivars.

Juice volatile composition differences between Valencia orange and its mutant Rohde Red Valencia are associated with carotenoid profile differences.

A targeted approach using HS-SPME-GC-MS was performed to investigate volatile compounds of ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV) at different developmental stages. Fifty-six volatile components classified into six chemical groups were quantified. The individual volatile compounds in each group were significantly influenced by the genotype and harvest date. Cluster analysis suggested two principal clusters. Cluster I included fruits of VAL and RRV harvested in July, September, and November and was characterized by the 2-ethyl-1-hexanol acetate and linalool chemotype; cluster II included fruits harvested in January and March, with the valencene/ß-myrcene chemotype. Principal component analysis confirmed that fruits of both cultivars harvested at different dates possessed a unique aroma active profile, especially, the odor-active volatile-norisoprenoids degraded from C40 carotenoid were significantly higher in RRV than in VAL at full maturity. These results could provide information for further study of the flavor metabolism pathways in the two closely related cultivars.

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.

Metabolically speaking: Possible reasons behind the tolerance of 'Sugar Belle' mandarin hybrid to huanglongbing.

Huanglongbing (HLB) is currently considered the most destructive disease of citrus. Since its spread to the Americas, HLB has killed millions of trees and caused a sharp decline in production in many citrus growing regions. With the continuous spread of HLB disease in Florida and worldwide, there is an urgent need for the development of commercial citrus cultivars with a strong tolerance to HLB. Interestingly, field observations showed that some of the recently released mandarin hybrids such as 'Sugar Belle' were tolerant to HLB. In this study, we investigated the volatile and non-volatile metabolites of greenhouse-grown 'Sugar Belle' mandarin and four of its ancestors in order to understand why 'Sugar Belle' mandarin is relatively tolerant to HLB. Leaf volatiles were directly extracted with hexane and analyzed using gas chromatography-mass spectrometry (GC-MS). Leaf polar metabolites were extracted with a mixture of methanol:water (1:1, v/v), derivatized to their trimethylsilyl ethers, and analyzed using GC-MS. Forty-seven volatile compounds and forty-two polar metabolites were detected in 'Sugar Belle' mandarin leaves and its ancestors. 'Sugar Belle' was high in several volatiles such as α-thujene, para-cymene, γ-terpinene, thymol, ß-elemene, and (E)-ß-caryophyllene. Some of these volatiles, especially thymol, ß-elemene, and (E)-ß-caryophyllene are known for their anti-microbial activity. In addition, 'Sugar Belle' mandarin was the highest in synephrine, benzoic acid, ferulic acid, caffeic acid, chiro-inositol, fructose, glucose, threonic acid, saccharic acid, and galactaric acid, and the second in threonine, malic acid, and myo-inositol compared to the ancestors. Phenolic compounds such as benzoic, ferulic, and caffeic acids may act as antibacterial agents, whereas others like sugar alcohols may protect 'Sugar Belle' mandarin from stress during pathogen attack. The tolerance of 'Sugar Belle' and other newly released mandarin hybrids should be further evaluated using greenhouse controlled studies. If tolerance of these hybrids is confirmed, they could be used to replace the traditionally susceptible cultivars.

Effect of Low-Furanocoumarin Hybrid Grapefruit Juice Consumption on Midazolam Pharmacokinetics.

The objectives of this study were to investigate the effect of grapefruit juice low in furanocoumarins on CYP3A activity and to summarize previous findings of enzyme inhibition measured by the metabolism of midazolam after intake of grapefruit juice. Twelve healthy volunteers participated in a prospective, randomized, double-blinded, 3-way crossover clinical study to determine the effect of regular grapefruit juice (RGJ) and a novel, low-furanocoumarin hybrid grapefruit juice (HGJ) on the metabolism of oral midazolam, used as a probe for in vivo CYP3A activity, compared with water as a control. The RGJ was 100% hand-squeezed "Hudson" grapefruit juice, and the HGJ contained low amounts of furanocoumarin constituents. The point estimates (90% confidence intervals) for the RGJ/water midazolam AUC geometric mean ratio was 122% (107-140). The point estimate for the HGJ/water midazolam AUC ratio was within the 80% to 125% bioequivalence range, indicating an absence of interaction. This finding also prompted a systematic review of available evidence on the pharmacokinetic alteration of midazolam by grapefruit juice. Although most studies demonstrated alteration in midazolam pharmacokinetics supporting inhibition of CYP3A activity as a likely mechanism, the cohorts included in these studies and the extent of the pharmacokinetic interaction varied widely. The current study indicated grapefruit juice-drug interaction varies substantially based on patient characteristics and/or grapefruit juice product-related factors, including the amount of furanocoumarin constituents present in the juice.

Construction of citrus gene coexpression networks from microarray data using random matrix theory.

After the sequencing of citrus genomes, gene function annotation is becoming a new challenge. Gene coexpression analysis can be employed for function annotation using publicly available microarray data sets. In this study, 230 sweet orange (Citrus sinensis) microarrays were used to construct seven coexpression networks, including one condition-independent and six condition-dependent (Citrus canker, Huanglongbing, leaves, flavedo, albedo, and flesh) networks. In total, these networks contain 37 633 edges among 6256 nodes (genes), which accounts for 52.11% measurable genes of the citrus microarray. Then, these networks were partitioned into functional modules using the Markov Cluster Algorithm. Significantly enriched Gene Ontology biological process terms and KEGG pathway terms were detected for 343 and 60 modules, respectively. Finally, independent verification of these networks was performed using another expression data of 371 genes. This study provides new targets for further functional analyses in citrus.

Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease.

BACKGROUND: Huanglongbing (HLB), the most devastating disease of citrus, is associated with infection by Candidatus Liberibacter asiaticus (CaLas) and is vectored by the Asian citrus psyllid (ACP). Recently, the molecular basis of citrus-HLB interactions has been examined using transcriptome analyses, and these analyses have identified many probe sets and pathways modulated by CaLas infection among different citrus cultivars. However, lack of consistency among reported findings indicates that an integrative approach is needed. This study was designed to identify the candidate probe sets in citrus-HLB interactions using meta-analysis and gene co-expression network modelling. RESULTS: Twenty-two publically available transcriptome studies on citrus-HLB interactions, comprising 18 susceptible (S) datasets and four resistant (R) datasets, were investigated using Limma and RankProd methods of meta-analysis. A combined list of 7,412 differentially expressed probe sets was generated using a Teradata in-house Structured Query Language (SQL) script. We identified the 65 most common probe sets modulated in HLB disease among different tissues from the S and R datasets. Gene ontology analysis of these probe sets suggested that carbohydrate metabolism, nutrient transport, and biotic stress were the core pathways that were modulated in citrus by CaLas infection and HLB development. We also identified R-specific probe sets, which encoded leucine-rich repeat proteins, chitinase, constitutive disease resistance (CDR), miraculins, and lectins. Weighted gene co-expression network analysis (WGCNA) was conducted on 3,499 probe sets, and 21 modules with major hub probe sets were identified. Further, a miRNA nested network was created to examine gene regulation of the 3,499 target probe sets. Results suggest that csi-miR167 and csi-miR396 could affect ion transporters and defence response pathways, respectively. CONCLUSION: Most of the potential candidate hub probe sets were co-expressed with gibberellin pathway (GA)-related probe sets, implying the role of GA signalling in HLB resistance. Our findings contribute to the integration of existing citrus-HLB transcriptome data that will help to elucidate the holistic picture of the citrus-HLB interaction. The citrus probe sets identified in this analysis signify a robust set of HLB-responsive candidates that are useful for further validation.

Surface barriers of mandarin 'okitsu' leaves make a major contribution to canker disease resistance.

Field evaluations have shown that Satsuma mandarin (Citrus unshiu) 'Okitsu' is one of the mandarin cultivars that shows substantial resistance to Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus bacterial canker disease. However, the mechanisms underlying this resistance are not well understood. In this study, we have shown that 'Okitsu' leaves are nevertheless susceptible to X. citri infection during a period of their development; however, this period is shorter than that seen in the susceptible mandarin 'Clemenules' (C. clementina). Under controlled growth conditions, the resistance of 'Okitsu' to X. citri was associated with the age of the leaf and was evident in spray-inoculated plants but not in those inoculated by infiltration. Furthermore, X. citri showed reduced attachment and biofilm formation in 'Okitsu' leaves compared with 'Clemenules'. Taken together, our data suggest that structural features of the 'Okitsu' leaf surface, such as the physical properties of the cuticle, are involved in the resistance to X. citri.