Identification of RNA silencing suppressor encoded by citrus chlorotic dwarf-associated virus

IntroductionCitrus chlorotic dwarf-associated virus (CCDaV) is an economically important citrus virus associated with leaf curling, deformation, and chlorosis found in China. Plants have evolved RNA silencing to defend against viral infections; however, the mechanism by which CCDaV suppresses RNA silencing in citrus remains unknown.MethodsSix proteins encoded by CCDaV were ectopically expressed in Nicotiana benthamiana 16c using the pCHF3 vector to identify RNA-silencing suppression activities.ResultsV2 protein encoded by CCDaV suppressed local RNA silencing and systemic RNA silencing triggered by GFP RNA, but did not impede short-distance movement of the RNA silencing signal in N. benthamiana 16c. GFP fluorescence observations showed that the ability of V2 protein to suppress RNA silencing was weaker than tomato bushy stunt virus P19. Deletion analysis showed that the putative nuclear localization signal (NLS, 25–54 aa) was involved in the RNA silencing suppression activity of V2 protein. Furthermore, V2 protein cannot block dsRNA-triggered RNA silencing. The subcellular localization assay suggested that V2 protein was localized to nucleus of N. benthamiana.ConclusionOverall, the results of this study demonstrate that CCDaV-V2 acts as an activity of silencing suppression. This is the first reported RNA-silencing suppressor encoded by Citlodavirus and will be valuable in revealing the molecular mechanism of CCDaV infection

The Complete Genome Sequence of ‘Candidatus Liberibacter solanacearum’, the Bacterium Associated with Potato Zebra Chip Disease

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with ‘Candidatus Liberibacter solanacearum’, a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for ‘Ca. L. solanacearum’. Here we present the sequence of the 1.26 Mbp metagenome of ‘Ca. L. solanacearum’, based on DNA isolated from potato psyllids. The coding inventory of the ‘Ca. L. solanacearum’ genome was analyzed and compared to related Rhizobiaceae to better understand ‘Ca. L. solanacearum’ physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, ‘Ca. L. solanacearum’ is related to ‘Ca. L. asiaticus’, a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to ‘Ca. L. asiaticus’, ‘Ca. L. solanacearum’ probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes

Optimizing the YOLOv7-Tiny Model with Multiple Strategies for Citrus Fruit Yield Estimation in Complex Scenarios

The accurate identification of citrus fruits is important for fruit yield estimation in complex citrus orchards. In this study, the YOLOv7-tiny-BVP network is constructed based on the YOLOv7-tiny network, with citrus fruits as the research object. This network introduces a BiFormer bilevel routing attention mechanism, which replaces regular convolution with GSConv, adds the VoVGSCSP module to the neck network, and replaces the simplified efficient layer aggregation network (ELAN) with partial convolution (PConv) in the backbone network. The improved model significantly reduces the number of model parameters and the model inference time, while maintaining the network’s high recognition rate for citrus fruits. The results showed that the fruit recognition accuracy of the modified model was 97.9% on the test dataset. Compared with the YOLOv7-tiny, the number of parameters and the size of the improved network were reduced by 38.47% and 4.6 MB, respectively. Moreover, the recognition accuracy, frames per second (FPS), and F1 score improved by 0.9, 2.02, and 1%, respectively. The network model proposed in this paper has an accuracy of 97.9% even after the parameters are reduced by 38.47%, and the model size is only 7.7 MB, which provides a new idea for the development of a lightweight target detection model

Differentially expressed genes involved in starch metabolism (A) and photosynthesis (B).

<p>Colored squares indicate up- or down-regulated genes with log₂ fold change (FC) ≥ 1.00 or ≤ -1.00.</p

Gene ontology classification of differentially expressed genes in different citrus hosts in response to ‘<i>Candidatus</i> Liberibacter asiaticus’.

<p>BP, Biological Process; CC, Cell Component; MF, Molecular Function.</p

Comparative PageMan display of perturbed pathways in <i>C</i>Las-affected <i>C</i>. <i>hystrix</i> and <i>C</i>. <i>sinensis</i>.

<p>The log₂ fold change of gene expression (mock-inoculated controls versus CLas-inoculated plants) was input into PageMan and subjected to a Wilcoxon test. Results were shown as a false-color heat-map-like display. Significantly up-regulated pathways are colored in red, while those colored in green are significantly down-regulated. Pathways without significant changes are white. Names of pathways are indicated on the right panel. CH, CH-M-VS-CH-HLB; CS, CS-M-VS-CS-HLB.</p

Statistic of differentially expressd genes (DEGs) of different citrus cultivars in response to <i>C</i>Las.

<p>CH, <i>Citrus hystrix</i>; CS, <i>C</i>. <i>sinensis</i>; M, Mock/healthy; HLB, Huanglongbing; CH-M-VS-CH-HLB, DEGs in HLB-infected <i>C</i>. <i>hystrix</i> compared with healthy control; CS-M-VS-CS-HLB, DEGs in HLB-infected <i>C</i>. <i>sinensis</i> compared with healthy control; CS-M-VS-CH-M, DEGs between healthy <i>C</i>. <i>hystrix</i> and healthy <i>C</i>. <i>sinensis</i>; CS-HLB-VS-CH-HLB, DEGs between HLB-infected <i>C</i>. <i>hystrix</i> and HLB-infected <i>C</i>. <i>sinensis</i>.</p

Comparative transcriptome analysis unveils the tolerance mechanisms of <i>Citrus hystrix</i> in response to ‘<i>Candidatus</i> Liberibacter asiaticus’ infection

<div><p>Citrus Huanglongbing (HLB), a highly devastating citrus disease, is associated with ‘<i>Candidatus</i> Liberibacter asiacitus’ (<i>C</i>Las), a member of phloem-inhabiting <i>α-proteobacteria</i>. HLB can affect all cultivated citrus and no cure is currently available. Previous studies showed that Kaffir lime (<i>Citrus hystrix</i>), primarily grown in South Asia and Southeast Asia, was tolerant to HLB but the molecular mechanism remains unknown. In this study, gene expression profiling experiments were performed on HLB-tolerant <i>C</i>. <i>hystrix</i> and HLB-susceptible <i>C</i>. <i>sinensis</i> three months after inoculation with <i>C</i>Las using RNA-seq data. Differentially expressed genes (DEGs) in the two citrus cultivars were mainly involved in diverse cellular functions including carbohydrate metabolism, photosynthesis, cell wall metabolism, secondary metabolism, hormone metabolism and oxidation/reduction processes. Notably, starch synthesis and photosynthesis process were not disturbed in <i>C</i>Las-infected <i>C</i>. <i>hystrix</i>. Most of the DEGs involved in cell wall metabolism and secondary metabolism were up-regulated in <i>C</i>. <i>hystrix</i>. In addition, the activation of peroxidases, Cu/Zn-SOD and POD4, may also enhance the tolerance of <i>C</i>. <i>hystrix</i> to <i>C</i>Las. This study provides an insight into the host response of HLB-tolerant citrus cultivar to <i>C</i>Las. <i>C</i>. <i>hystrix</i> is potentially useful for HLB-tolerant/resistant citrus breeding in the future.</p></div

MapMan analysis of differentially expressed genes in <i>C</i>Las-affected <i>Citrus hystrix</i> (A) and <i>C</i>Las-affected <i>C</i>. <i>sinensis</i> (B) involved in stress responses.

<p>Red squares represent genes those were significantly up-regulated; green squares represent genes those were significantly down-regulated.</p

RT-qPCR and RNA-seq profiles of 16 selected differentially expressed genes (DEGs).

<p>A. DEGs between healthy and HLB-infected <i>Citrus sinensis</i>; B, DEGs between healthy and HLB-infected <i>C</i>. <i>hystrix</i>; C, DEGs between HLB-infected <i>C</i>. <i>hystrix</i> and HLB-infected <i>C</i>. <i>sinensis</i>. glgA, starch synthase; TPP, trehalose 6-phosphate phosphatase; LHCb1, light-harvesting complex II chlorophyll a/b binding protein 1; PAL, phenylalanine ammonia-lyase-like; 2OG-Fe(II), 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase; CESA, cellulose synthase A; XTH23, xyloglucan endotransglucosylase/hydrolase protein 23; PHT, phosphate transporter; ZIP2, zinc transporter 2; EREBP, ethylene-responsive transcription factor ERF017; SODC, superoxide dismutase [Cu-Zn]; LRR-1, LRR receptor-like serine/threonine-protein kinase; LRR-2, LRR-like serine/threonine-protein kinase BAM2; RGA3, disease resistance protein RGA3, NB-ARC class; EDS1 (enhanced disease susceptibility 1 protein); MIR, miraculin.</p