field-deployable-candidatus-liberibacter-asiaticus-detection-using

Field-Deployable <em>Candidatus</em> Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A ...

Gannan Normal University

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the transmitting vector of Candidatus Liberibacter asiaticus (CLas), which causes citrus disease Huanglongbing (HLB). In recent years, control of HLB has been achieved by reducing the vector population. In the present study, we identified an isoform of D. citri tropomyosin (herein designated as DcTm1-X1). DcTm1-X1 was down-regulated in CLas-infected ACPs compared with uninfected ACPs. Bioinformatics analysis revealed that the full-length DcTm1-X1 is 2955 bp and encodes a protein of 284 amino acids with a deduced molecular weight of 32.15 kDa. Phylogenetic tree analysis suggested that DcTm1-X1 shares a high amino acid identity with its homolog in Acyrthosiphon pisum. Higher DcTm1-X1 expression levels were found in the leg of the psyllid by reverse transcription quantitative PCR (RT-qPCR). According to Blue Native PAGE analysis and mass spectrometric analysis, DcTm1-X1 interacts with citrate synthase (CS) and V-type proton ATPase subunit B-like (VAT). In addition, knockdown of DcTm1-X1 by RNA interference (RNAi) significantly increased the mortality rate of nymphs and the infection rate of CLas at different time points. Taken together, our results show that DcTm1-X1 might play an important role in response to CLas, but also lay a foundation for further research on the functions of DcTm1-X1.

Potential roles of insect Tropomyosin1-X1 isoform in the process of Candidatus Liberibacter asiaticus infection of Diaphorina citri.

Chitin synthase is a critical enzyme that catalyzes -acetylglucosamine to form chitin, which plays an important role in the growth and development of insects. In this study, we identified a chitin synthase gene () with a complete open reading frame (ORF) of 3180 bp from the genome database of , encoding a protein of 1059 amino acid residues with the appropriate signature motifs (EDR and QRRRW). Reverse transcription-quantitative PCR (RT-qPCR) analysis suggested that  () was expressed throughout all developmental stages and all tissues.  had the highest expression level in the integument and fifth-instar nymph stage. Furthermore, the effects of diflubenzuron (DFB) on  mortality and  expression level were investigated using fifth-instar nymph through leaf dip bioassay, and the results revealed that the nymph exposed to DFB had the highest mortality compared with control group (Triton-100). Silencing of  by RNA interference resulted in malformed phenotypes and increased mortality with decreased molting rate. In addition, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) also revealed corresponding ultrastructural defects. Our results suggest that  might play an important role in the development of  and can be used as a potential target for psyllid control.

Silencing of the Chitin Synthase Gene Is Lethal to the Asian Citrus Psyllid, .

Seven isolates of a putative cytorhabdovirus (family , order ) designated as citrus-associated rhabdovirus (CiaRV) were identified in citrus, passion fruit, and paper bush from the same geographical area in China. CiaRV, bean-associated cytorhabdovirus (Brazil), and papaya virus E (Ecuador) should be taxonomically classified in the species . Due to natural mutations, the glycoprotein (G) and P4 genes were impaired in citrus-infecting isolates of CiaRV, resulting in an atypical rhabdovirus genome organization of 3' leader-N-P-P3-M-L-5' trailer. The P3 protein of CiaRV shared a common origin with begomoviral movement proteins (family ). Secondary structure analysis and -complementation of movement-deficient tomato mosaic virus and potato virus X mutants by CiaRV P3 supported its function in viral cell-to-cell trafficking. The wide geographical dispersal of CiaRV and related viruses suggests an efficient transmission mechanism, as well as an underlying risk to global agriculture. Both the natural phenomenon and experimental analyses demonstrated presence of the "degraded" type of CiaRV in citrus, in parallel to "undegraded" types in other host plant species. This case study shows a plant virus losing the function of an important but nonessential gene, likely due to host shift and adaption, which deepened our understanding of course of natural viral diversification.

Natural Defect of a Plant Rhabdovirus Glycoprotein Gene: A Case Study of Virus-Plant Coevolution.

The black citrus aphid  is a major pest of citrus and tea plants. In this study, we determined the complete mitochondrial genome (mitogenome) of . The mitogenome was 15,469 bp in length, consisting of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding control region. Gene order of  was highly conserved and identical to most other previously sequenced Aphididae. Initiation codons ATT, ATA, and ATG were identified in eight, three, and two PCGs, respectively, while stop codon TAA was found in 11 genes except for 2 genes ( and ) which use incomplete stop codon T-. Phylogenetic analysis showed the topology (Pemphigidae + (Hormaphididae + (Greenideidae + Aphididae))) within Aphidoidea, and  was more closely related to  than to .

Phylogenetic relationship and characterization of the complete mitochondrial genome of the black citrus aphid,  (Hemiptera: Aphididae).

 (Odonata: Aeshnidae) is a big dragonfly which can be seen patrolling around ponds, lakes and other still water. In this study, we sequenced and analyzed the complete mitochondrial genome (mitogenome) of . This mitogenome was 15,366 bp long and encoded 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA unit genes (rRNAs). The nucleotide composition of the mitogenome was biased toward A and T, with 74.8% of A + T content (A 40.1%, T 34.7%, C 14.0%, G 11.2%). Gene order was conserved and identical to most other previously sequenced Aeshnidae dragonflies. Most PCGs of  have the conventional start codons ATN (six ATG, three ATT, and two ATC), with the exception of  and  (TTG). Except for three genes (, , and ) end with the incomplete stop codon T--, all other PCGs terminated with the stop codon TAA. Phylogenetic analysis showed that  is sister to  with high support value. All 15 Anisoptera species constituted a major clade with well support, and Aeshnidae had a close relationship with Gomphidae and Libellulidae.

Complete mitochondrial genome sequence of  (Odonata: Anisoptera: Aeshnidae) and phylogenetic analysis.

 is a commonly seen dragonfly with a big yellow or white ringlike spot on the third and fourth segments of its abdomen. In this study, we sequenced and analyzed the complete mitochondrial genome (mitogenome) of . This mitogenome was 15,434 bp long and encoded 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and 2 ribosomal RNA unit genes (rRNAs). Gene order was conserved and identical to most other previously sequenced Libellulidae dragonflies. The whole mitogenome exhibited heavy AT nucleotide bias (74.6%). Most PCGs of  have the conventional start codons ATN (six ATG, three ATT, and two ATC), with the exception of  and  (TTG). Except for four genes (, , , and ) end with the incomplete stop codon T-, all other PCGs terminated with the stop codon TAA or TAG. Phylogenetic analysis showed that  got together with  with high support value, and the relationships (( + ) + (( + ) + ( + ))) were supported in Libellulidae.

Article	Year
Potential roles of insect Tropomyosin1-X1 isoform in the process of Candidatus Liberibacter asiaticus infection of Diaphorina citri. Journal of insect physiology\| PubMed ID: 30817914	2019
Silencing of the Chitin Synthase Gene Is Lethal to the Asian Citrus Psyllid, . International journal of molecular sciences\| PubMed ID: 31370145	2019
Natural Defect of a Plant Rhabdovirus Glycoprotein Gene: A Case Study of Virus-Plant Coevolution. Phytopathology\| PubMed ID: 32648524	2021
Phylogenetic relationship and characterization of the complete mitochondrial genome of the black citrus aphid, (Hemiptera: Aphididae). Mitochondrial DNA. Part B, Resources\| PubMed ID: 33366088	2019
Complete mitochondrial genome sequence of (Odonata: Anisoptera: Aeshnidae) and phylogenetic analysis. Mitochondrial DNA. Part B, Resources\| PubMed ID: 33490598	2021
Characterization and phylogenetic analysis of the complete mitochondrial genome of (Odonata: Anisoptera: Libellulidae). Mitochondrial DNA. Part B, Resources\| PubMed ID: 33521259	2021

Aijun Huang

Biography

JoVE Journal Publications

Other Publications