Glucose-1-phosphate adenylyltransferase was highly down-regulated in symptomatic fruits while invertase was clearly up-regulated in the peel of apparently healthy and symptomatic fruits from the infected orchard. Sucrose symporter was up-regulated in infected, symptomatic fruits. The terpenoid pathway was affected by HLB disease, as shown by the down-regulation of terpene synthase cyclase . Induction of lipid transfer protein was observed in the absence of symptoms. The expression of other genes such as acidic cellulase and methyltransferase2 was diminished in infected fruits.Huanglongbing, a highly destructive disease of citrus, threatens citrus-producing areas worldwide. Previous studies monitored transcriptional changes in leaves on a large scale using microarrays to investigate host responses and reveal the mechanisms underlying disease development. The present work focuses on transcriptional regulation in fruit peel to analyze the response to CaLas infection at different disease stages. Next-generation sequencing technology , as used here, can find differential expression of an increased number of transcripts, many of which may not be present in EST databases or represented in microarrays. NGS data can be used for specific transcriptome assemblies that become resource datasets for annotation of genomes and annotation of differentially regulated genes and proteins analyzed using any ‘‘omic’’ technique. However, 30 planter pot the absence of a completed genome sequence limited the advantages of RNA-Seq technologies. The aim of this work was to provide data using NGS technology for a comprehensive analysis of metabolic changes in fruit induced by HLB disease.

These findings will uncover the fruit disorder mechanisms and facilitate development of short-term therapeutic strategies for already-infected trees. Toward this end, the experimental design included four types of fruit: healthy control fruit from an HLB-free orchard, apparently healthy fruit from non-symptomatic trees in an orchard affected by HLB, and asymptomatic and symptomatic fruit from infected symptomatic trees in the affected orchard. This design made it possible to identify differentially expressed genes at different disease stages. A comparison between apparently healthy and asymptomatic fruit revealed genes induced early in disease development. Comparing asymptomatic and symptomatic fruit identified genes involved in the host response during disease progression. Comparing symptomatic and apparently healthy fruit revealed host response genes related to the presence of HLB symptoms. Healthy fruit from the HLB-free location were also compared to the three sample types from the infected location. Differences in gene expression in these comparisons are likely to result from environmental and agronomic variability due to the difference in location, in addition to the effects of HLB. A range of 1154 to 1762 differentially regulated genes were found using RNA-Seq comparing the three categories of fruits from the infected orchard with those taken from a location free of HLB . In qRTPCR analysis, 31 of 33 differentially regulated genes confirmed the pattern of expression found by RNA-Seq. Comparisons among the three fruit categories in the infected orchard resulted in fewer differentially regulated genes . This was expected, since samples within the same orchard grew under similar environmental and agronomic conditions. A similar contrast between samples from the infected and uninfected orchards is seen in principal component analysis. The transcripts that most strongly characterize the asymptomatic and apparently healthy samples are common between the two samples.

The symptomatic and uninfected orchard samples are both distinct groups and widely separated in the biplot . A visual summary outlines the most important transcriptional changes in the networks among genes, pathways, and cell functions in the fruit peel . Gene set enrichment analysis identified several pathways significantly affected by HLB as symptoms appear, considering both within-tree and between-tree comparisons, such as those for phenylpropanoids, starch and sucrose metabolism, carbon fixation, ascorbate, and alpha-linolenic acid . Other pathways were also affected by HLB in the two comparisons within the same orchard. Transcripts encoding different subunits of the photosystem II reaction center and cytochrome b6-f complex subunit were more abundant in symptomatic HLB-infected fruit than in healthy fruit. Photosynthesis is central to all aspects of plant biology, since it provides energy for growth and reproduction, but its regulation by biotic and abiotic stresses is still unclear. The induction of photosynthetic light reactions in the fruit is consistent with the observation that symptomatic HLB-infected fruit often remains green. The retention of green color and increase of photosynthesis reactions is probably linked to the lower amount of ethylene detected insymptomatic fruits. Photosynthesis is usually downregulated by pathogen attacks. The upregulation of photosynthetic reactions in the fruit does not contradict this common consideration because CaLas infections typically occur in young leaves. The transcriptomic changes observed in the fruit are probably linked to the source-sink disruption caused by leaf infections. Protein degradation and modification pathways were significantly changed by CaLas infection, as shown by the upregulation of genes such as C3HC4-type ring finger proteins involved in ubiquitin-mediated degradation. Interestingly, heat shock proteins HSP82 and HSP70, highly interactive proteins in the PPI network inferred in citrus, were down-regulated at different stages of the disease. Heat shock proteins are highly conserved proteins induced in cells subjected to elevated temperatures or other environmental stresses.

These proteins act as molecular chaperones to stabilize, reduce misfolding, or facilitate refolding of proteins that have been denatured during stress events. In plant cells, HSP70 and HSP90 are involved in signal transduction leading to plant defense responses. Both proteins interact with a salicylic acid-induced protein kinase and their silencing affected the hypersensitive response in Nicotiana benthamiana while inducing non-host resistance to Phytophthora infestans. HSP90 also modulates the innate immune responses involving gene-for-gene specific interactions, acting as a scaffold protein in a complex that mediates signal transduction. Based on these findings, we speculate that down-regulation of heat shock proteins observed at different stages of HLB disease might increase protein misfolding in the fruit peel. Genes encoding ATP synthase gamma and delta chains were also induced in symptomatic fruit, supporting the idea that CaLas may act as an energy parasite by scavenging ATP from its host with a pathogen-specific ATP/ADP translocase. Indeed, the recently sequenced genome of CaLas revealed the presence of an ATP/ADP translocase in addition to ATP synthase. ATP scavenging may be a possible mechanism of pathogenicity, affecting the fruit peduncle, columella, and seed coat. Photosynthesis regulation in infected fruit peel may also reduce transport of sugars and ions such as nitrate, sulfate, and potassium. In this study, several differentially expressed genes were associated with transport of ions, including ammonium, sulfate, and phosphate . Inorganic ions can modify sugar metabolism and photosynthesis. Polarized vesicle trafficking, transport, and secretion of plant materials are associated primarily with non-specific resistance during host-pathogen interactions. ABC transporters play important roles in this process, since they are also involved in virulence, host range, and symptom elicitation. Citrus proteins related to sugar and PDR/ABC transporters implicated in secretion of antimicrobial terpenoids were specifically induced by HLB at the symptomatic stage . Interestingly, among the genes identified in the CaLas genome were those for phosphate and zinc uptake into the cell. This implies that mineral uptake by the pathogen may be enhanced due to induction of endogenous genes as well as host genes. However, plastic growers pots phloem necrosis induced by CaLas contributes to the impaired nutritional transport functions and source-sink communication observed in this study. Starch accumulation in leaf chloroplasts of sweet orange trees infected with CaLas has previously been demonstrated and is characteristic for HLB. Genes encoding the large subunit of ADP-glucose pyrophosphorylase, the key enzyme catalyzing the first and limiting step in starch biosynthesis, were up-regulated in infected leaves. Interestingly, a gene encoding the large subunit of glucose-1-phosphate adenylyltransferase was downregulated in infected and symptomatic fruit in the present study. Also of interest are contrasting patterns of expression for different isoforms of starch synthase and starch cleavage genes, leaving the dynamics of starch accumulation in the fruit unclear . This is in contrast to experimental observation of starch accumulation in infected leaf tissues. In leaves, this accumulation was linked to up-regulation of genes involved in starch biosynthesis and down-regulation of its conversion to maltose. It has been hypothesized that starch accumulation is an effect of phloem plugging/necrosis during HLB infection, although it usually occurs before these symptoms are visible. Differences in starch accumulation are probably linked to the different types of organs: mature leaves are ‘‘source’’ and fruit are ‘‘sink’’. However, further analysis of fruit starch accumulation must be performed to validate this hypothesis.

An increased abundance of transcripts for genes involved in the first steps of glycolysis and sucrose metabolism was observed in fruits from infected trees. In the cytosol, an invertase gene was upregulated in asymptomatic and symptomatic fruit, affecting the sugar balance and communication between sink and source tissues. We speculate that this leads to increased glucose and fructose and decreased sucrose in fruit cells, which should be further validated with carbohydrate analysis. It is important to note that elevated glucose and fructose has been demonstrated in citrus leaves infected with CaLas. Interestingly, different types of invertases were up-regulated in leaves and fruits . Over-expression of yeast invertase in the cell wall of transgenic tobacco disrupted sucrose export, allowing soluble sugars and starch to accumulate, which consequently inhibited photosynthesis and resulted in stunted growth and bleached or necrotic leaf areas. As previously suggested for leaves, it is possible that the differential expression of key genes involved in sucrose and starch metabolism, as observed in CaLas-infected citrus fruit, might affect the osmotic potential and induce plasmolysis, thus altering the ripening process and producing typical HLB symptoms. However, further analysis of sugar concentrations will be necessary to clarify the causes and effects of disease symptoms in the fruit. Other studies on CaLas-infected leaves have shown increased sucrose and glucose, but not fructose. In fruit, it is possible that altered fructose and glucose concentrations might be responsible for physiological disorders and affect source-sink relationships with leaves. The gene set enrichment analysis confirmed that sucrose and starch metabolism were highly affected by the disease. Integrated analysis of leaf and fruit data indicates that sugar and starch metabolism play a key role in the metabolic dysfunction induced by HLB disease. However, few studies have been conducted to address the effects of altering sugar metabolism on resistance to pathogen infections. Invertase plays a key role in the activation of stress responses and may function as an extracellular indicator for pathogen infection. Indeed, transgenic plants overexpressing sugar metabolism enzymes such as a heterologous invertase from yeast have helped clarify source-sink relationships. The expression of different viral movement proteins in transgenic plants and the resulting effects on photosynthesis, carbohydrate accumulation, and partitioning emphasize the importance of sugars in activating defense responses against biotic attacks. Sink metabolism may be essential to satisfy the energy requirements of activating the cascade of defense responses. Interestingly, sucrose synthase was also more abundant in symptomatic fruit while sucrose transporter genes were downregulated by HLB. The Genevestigator database, indicated that these proteins may be down-regulated by hormones such as ethylene, methyl-jasmonates, and indol-3-acetic acid. The concept of sucrose metabolism regulators as a potential target for HLB therapeutics is intriguing. Increasing evidence indicates an extensive cross-talk between sugar, hormone, and light signal transduction networks in plants. Hormone pathways were significantly altered in fruit peel inresponse to CaLas infection. Two genes involved in auxin synthesis, GH3.1 and GH3.4, were induced in affected fruit. Gibberellin regulation has been observed in other fruit disorders such as albedo breakdown disorder and applications of GA3 before fruit color break can reduce the occurrence of some fruit disorders. It is possible that sugar metabolism changes observed in the fruit might be linked with the down-regulation of gibberellins that regulate energy and carbohydrate metabolism. Previous studies have demonstrated that regulation of gibberellic acid-induced gene expression is affected by sugar and hormone signaling. That cytokinins play a role in sugar regulation has been demonstrated. Therapeutic approaches using small-molecule hormones such as cytokinins and gibberellins may allow modification of fruit metabolism to mitigate the negative impact of HLB on fruit quality and productivity.Ethylene regulates a variety of developmental processes and stress responses in plants, including seed germination, cell elongation, senescence, fruit ripening, and defense. Nonetheless, ethylene can promote either disease resistance or susceptibility, depending on the host–pathogen interaction. In our study, considerable changes were observed in the transcriptional profiles of genes related to ethylene biosynthesis and signal transduction. ACC synthase and ACC oxidase play pivotal roles in ethylene biosynthesis and their expression is often affected by pathogen attack. It was unclear how ethylene concentration changes in fruit in response to HLB.