scholarly journals Not Just a Cycle: Three gab genes Enable the Non-cyclic Flux Toward Succinate Via GABA Shunt in ‘Candidatus Liberibacter asiaticus’-infected citrus

2021 ◽  
Author(s):  
Yasser Nehela ◽  
Nabil Killiny
Keyword(s):  
Tca Cycle ◽  
Defense Responses ◽  
Candidatus Liberibacter Asiaticus ◽  
Amino Acid Permease ◽  
Phytopathogenic Bacterium ◽  
Gaba Shunt ◽  
Semialdehyde Dehydrogenase ◽  
The Tca Cycle ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Although the mitochondria retain all required enzymes for an intact tricarboxylic acid (TCA) cycle, plants might shift the cyclic flux from the TCA cycle to an alternative non-cyclic pathway via γ-aminobutyric acid (GABA) shunt under specific physiological conditions. We hypothesize that several genes may ease this non-cyclic flux and contribute to the citrus response to the phytopathogenic bacterium ‘Candidatus Liberibacter asiaticus’, the causal agent of Huanglongbing in citrus. To test this hypothesis, we used multi-omics techniques (metabolomics, fluxomics, and transcriptomics) to investigate the potential role(s) of putative gab homologies from Valencia sweet orange (Citrus sinensis). Our findings showed that ‘Ca. L. asiaticus’ significantly increased the endogenous GABA and succinate content but decreased ketoglutarate in infected citrus plants. Citrus genome harbors three putative gab genes including amino-acid permease (aka GABA permease; CsgabP), GABA transaminase (CsgabT), and succinate-semialdehyde dehydrogenase (aka GABA dehydrogenase; CsgabD). The transcript levels of CsgabP, CsgabT, and CsgabD were upregulated in citrus leaves upon the infection with ‘Ca. L. asiaticus’ and after the exogenous application of GABA or deuterium-labeled GABA isotope (GABA-D6). Moreover, our finding showed that exogenously applied GABA is quickly converted to succinate and fed into the TCA cycle. Likewise, the fluxomics study showed that GABA-D6 is rapidly metabolized to succinate-D4. Our work proved that GABA shunt and three predicated gab genes from citrus, support the upstream non-cyclic flux toward succinate rather than an intact TCA cycle and contribute to citrus defense responses to ‘Ca. L. asiaticus’.

scholarly journals ‘Candidatus Liberibacter asiaticus’ and Its Vector, Diaphorina citri, Augment the Tricarboxylic Acid Cycle of Their Host via the γ-Aminobutyric Acid Shunt and Polyamines Pathway

2019 ◽  
Vol 32 (4) ◽  
pp. 413-427 ◽  
Author(s):  
Yasser Nehela ◽  
Nabil Killiny
Keyword(s):  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Aminobutyric Acid ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Gaba Shunt ◽  
The Tca Cycle ◽  
Candidatus Liberibacter ◽  
Nutritional Needs ◽  
Liberibacter Asiaticus

Huanglongbing (HLB), a destructive citrus disease, is associated with ‘Candidatus Liberibacter asiaticus’, which is transmitted by the Asian citrus psyllid Diaphorina citri. Both ‘Ca. L. asiaticus’ and its vector manipulate the host metabolism for their benefit, to meet their nutritional needs and neutralize the host defense responses. We used a targeted gas chromatography-mass spectrometry–based method to explore the connection between the tricarboxylic acid (TCA) cycle, γ-aminobutyric acid (GABA) shunt, and polyamines (PAs) pathways in citrus. ‘Ca. L. asiaticus’ and D. citri accelerated the conversion of α-ketoglutarate to glutamate, then to GABA, causing an accumulation of GABA in the cytosol. In silico analysis showed that the citrus genome possesses a putative GABA permease that connects the GABA shunt with the TCA cycle and supports the accumulation of succinate, fumarate, and citrate. Additionally, the PAs biosynthetic pathway might be connected directly to the TCA cycle, through the production of fumarate, or indirectly, via enhancement of GABA shunt. Taken together, we suggest that GABA shunt and PAs pathways are alternative pathways that contribute to the flux toward succinate rather than an intact TCA cycle in citrus. Both ‘Ca. L. asiaticus’ and its vector enhance these pathways. This study provides more insights into citrus responses to the HLB pathosystem and could be a further step toward clues for understanding the nutritional needs of ‘Ca. L. asiaticus’, which could help in culturing ‘Ca. L. asiaticus’.

Generous hosts: Growth of ‘Candidatus Liberibacter asiaticus’ in Madagascar periwinkle (Catharanthus roseus) highlights its nutritional needs

2021 ◽  
Author(s):  
Nabil Killiny
Keyword(s):  
Organic Acids ◽  
Catharanthus Roseus ◽  
Bacterial Growth ◽  
Tca Cycle ◽  
Candidatus Liberibacter Asiaticus ◽  
Madagascar Periwinkle ◽  
Phloem Sap ◽  
The Tca Cycle ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

‛Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening is not available in pure culture yet. In addition to trees of citrus and citrus relatives, ‛Ca. L. asiaticus’ can grow in Madagascar periwinkle (Catharanthus roseus). Using GC-MS, we compared the phloem sap composition in sweet orange ‛Valencia’ (Citrus sinensis) and periwinkle plants after the infection with ‛Ca. L. asiaticus’. Interestingly, in contrast to our previous studies of total leaf metabolites, we found that, compared to uninfected phloem sap, we found that the organic acids implicated in the TCA cycle including citrate, isocitrate, succinate, fumarate, and malate were reduced significantly in the infected phloem saps of both species. As a result of the reduction of organic acids content, the pH of infected phloem saps was increased. We hypothesize that the bacterial growth induces the mitochondrial TCA cycle in parenchyma cells to produce more of these compounds to be used as a bacterial carbon source. Once these compounds reach a low level in the phloem sap, the bacterium may send a signal, yet to be identified, to initiate a feedback loop to further induce the TCA cycle. Phloem blockage might be another reason behind the reduced the translocation of TCA cycle intermediates within the phloem. The net result, localized availability of organic acids likely benefits the bacterial growth and may explain the unequal distribution of ‘Ca L. asiaticus’ within infected trees. These findings may help in designing media for the pure culturing of ‛Ca. L. asiaticus’.

scholarly journals Diaphorina citri Genome Possesses a Complete Melatonin Biosynthesis Pathway Differentially Expressed under the Influence of the Phytopathogenic Bacterium, Candidatus Liberibacter asiaticus

Insects ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 317
Author(s):  
Yasser Nehela ◽  
Nabil Killiny
Keyword(s):  
Amino Acid ◽  
Shikimic Acid ◽  
Expression Patterns ◽  
Acid Decarboxylase ◽  
Biosynthesis Pathway ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Phytopathogenic Bacterium ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Melatonin is synthesized from the amino acid L-tryptophan via the shikimic acid pathway and ubiquitously distributed in both prokaryotes and eukaryotes. Although most of melatonin biosynthesis genes were characterized in several plants and animal species including the insect model, Drosophila melanogaster, none of these enzymes have been identified from the Asian citrus psyllid, Diaphorina citri. We used comprehensive in silico analysis and gene expression techniques to identify the melatonin biosynthesis-related genes of D. citri and to evaluate the expression patterns of these genes within the adults of D. citri with gradient infection rates (0, 28, 34, 50, 58, and 70%) of the phytopathogenic bacterium Candidatus Liberibacter asiaticus and after the treatment with exogenous melatonin. We showed that the D. citri genome possesses six putative melatonin biosynthesis-related genes including two putative tryptophan 5-hydroxylase (DcT5H-1 and DcT5H-2), a putative aromatic amino acid decarboxylase (DcAADC), two putative arylalkylamine N-acetyltransferase (DcAANAT-1 and DcAANAT-2), and putative N-acetylserotonin O-methyltransferase (DcASMT). The infection with Ca. L. asiaticus decreased the transcript levels of all predicted genes in the adults of D. citri. Moreover, melatonin supplementation induced their expression levels in both healthy and Ca. L. asiaticus-infected psyllids. These findings confirm the association of these genes with the melatonin biosynthesis pathway.

scholarly journals Identification of Gene Candidates Associated with Huanglongbing Tolerance, Using ‘Candidatus Liberibacter asiaticus’ Flagellin 22 as a Proxy to Challenge Citrus

2018 ◽  
Vol 31 (2) ◽  
pp. 200-211 ◽  
Author(s):  
Qingchun Shi ◽  
Vicente J. Febres ◽  
Shujian Zhang ◽  
Fahong Yu ◽  
Greg McCollum ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Citrus Canker ◽  
Defense Responses ◽  
Candidatus Liberibacter Asiaticus ◽  
Tolerance Mechanisms ◽  
Molecular Pattern ◽  
Number Of Genes ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

The 22–amino acid (flg22) pathogen-associated molecular pattern from the flagellin of Xanthomonas citri subsp. citri has been shown to induce defense responses correlated with citrus canker resistance. Here, flg22 of ‘Candidatus Liberibacter asiaticus’, the putative causal agent of Huanglongbing (HLB), elicited differential defense responses that were weaker than those from Xcc-flg22, between those of the HLB-tolerant mandarin cultivar Sun Chu Sha and susceptible grapefruit cultivar Duncan. Transcriptomics was used to compare the effect of CLas-flg22 and Xcc-flg22 between the citrus genotypes and identified 86 genes induced only by CLas-flg22 in the tolerant mandarin. Expression of 16 selected genes was validated, by reverse transcription-quantitative polymerase chain reaction, and was evaluated in citrus during ‘Ca. L. asiaticus’ infection. Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with ‘Ca. L. asiaticus’, suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and ‘Ca. L. asiaticus’ treatments, while others were oppositely regulated in the tolerant mandarin, suggesting similarity and interplay between CLas-flg22 and ‘Ca. L. asiaticus’–triggered defenses. Genes identified are valuable in furthering the study of HLB tolerance mechanisms and, potentially, for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy.

scholarly journals Two ‘Candidatus Liberibacter asiaticus’ Strains Recently Found in California Harbor Different Prophages

2017 ◽  
Vol 107 (6) ◽  
pp. 662-668 ◽  
Author(s):  
Z. Zheng ◽  
F. Wu ◽  
L. B. Kumagai ◽  
M. Polek ◽  
X. Deng ◽  
...  
Keyword(s):  
Los Angeles ◽  
Geographic Region ◽  
Mapping Technique ◽  
Polymorphism Analysis ◽  
Candidatus Liberibacter Asiaticus ◽  
Next Generation Sequencing Technology ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (CLas), an α-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, two cases of CLas have been detected in Los Angeles County, one in Hacienda Heights in 2012 and the other in San Gabriel in 2015. Although all infected trees were destroyed in compliance with a state mandate, citrus industry stakeholder concerns about HLB in California are high. Little is known about the biology of CLas, particularly the California strains, hindering effective HLB management efforts. In this study, next-generation sequencing technology (Illumina MiSeq) was employed to characterize the California CLas strains. Data sets containing >4 billion (Giga) bp of sequence were generated from each CLas sample. Two prophages (P-HHCA1-2 and P-SGCA5-1) were identified by the MiSeq read mapping technique referenced to two known Florida CLas prophage sequences, SC1 and SC2. P-HHCA1-2 was an SC2-like or Type 2 prophage of 38,989 bp in size. P-SGCA5-1 was an SC1-like or Type 1 prophage of 37,487 bp in size. Phylogenetic analysis revealed that P-HHCA1-2 was part of an Asiatic lineage within the Type 2 prophage group. Similarly, P-SGCA5-1 was part of an Asiatic lineage within Type 1 prophage group. The Asiatic relatedness of both P-HHCA1-2 and P-SGCA5-1 was further presented by single nucleotide polymorphism analysis at terL (encoding prophage terminase) that has been established for CLas strain differentiation. The presence of different prophages suggests that the two California CLas strains could have been introduced from different sources. An alternative explanation is that there was a mixed CLas population containing the two types of prophages, and limited sampling in a geographic region may not accurately depict the true CLas diversity. More accurate pathway analysis may be achieved by including more strains collected from the regions.

scholarly journals Susceptibility of Sixteen Citrus Genotypes to ‘Candidatus Liberibacter asiaticus’

Plant Disease ◽  
2016 ◽  
Vol 100 (6) ◽  
pp. 1080-1086 ◽  
Author(s):  
Greg McCollum ◽  
Mark Hilf ◽  
Mike Irey ◽  
Weiqi Luo ◽  
Tim Gottwald
Keyword(s):  
The United States ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Germplasm ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Florida Citrus ◽  
Citrus Macrophylla ◽  
Free Ranging

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

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