Integrated Analysis of the miRNAome and Transcriptome Reveals miRNA-mRNA Regulatory Networks in Catharanthus roseus Through Cuscuta campestris-Mediated Infection With "Candidatus Liberibacter asiaticus".

Chunhua Zeng,Xuefeng Wang,Changyong Zhou,Haodi Wu,Mengji Cao,Shimin Fu

doi:10.3389/fmicb.2022.799819

Abstract

Citrus Huanglongbing (HLB) is the most devastating disease of citrus caused by the Gram-negative phloem-limited bacterium “Candidatus Liberibacter asiaticus” (CLas). It can be transmitted by the Asian citrus psyllid “Diaphorina citri,” by grafting, and by the holoparasitic dodder. In this study, the non-natural host periwinkle (Catharanthus roseus) was infected via dodder (Cuscuta campestris) from CLas-infected citrus plants, and the asymptomatic leaves (AS) were subjected to transcriptomic and small-RNA profiling. The results were analyzed together with a transcriptome dataset from the NCBI repository that included leaves for which symptoms had just occurred (S) and yellowing leaves (Y). There were 3,675 differentially expressed genes (DEGs) identified in AS, and 6,390 more DEGs in S and further 2109 DEGs in Y. These DEGs were commonly enriched in photosystem, chloroplast, membrane, oxidation-reduction process, metal/zinc ion binding on GO. A total of 14,974 DEGs and 336 DE miRNAs (30 conserved and 301 novel) were identified. Through weighted gene co-expression network and nested network analyses, two critical nested miRNA–mRNA regulatory networks were identified with four conserved miRNAs. The primary miR164-NAC1 network is potentially involved in plant defense responses against CLas from the early infection stage to symptom development. The secondary network revealed the regulation of secondary metabolism and nutrient homeostasis through miR828-MYB94/miR1134-HSF4 and miR827-ATG8 regulatory networks, respectively. The findings discovered new potential mechanisms in periwinkle–CLas interactions, and its confirmation can be done in citrus–CLas system later on. The advantages of periwinkle plants in facilitating the quick establishment and greater multiplication of CLas, and shortening latency for disease symptom development make it a great surrogate for further studies, which could expedite our understanding of CLas pathogenesis.

Highlights

Citrus Huanglongbing (HLB) is one of the most destructive globally distributed citrus diseases and is responsible for tremendous economic losses to citrus industries worldwide (Wang, 2019)
40–47 million clean reads were obtained from each plant with/without Candidatus Liberibacter asiaticus” (CLas) infection, and approximately 56–60% of reads were successfully mapped to the C. roseus genome (Supplementary Table 2)
The results indicated that more genes and corresponding pathways were perturbed as symptom development

Summary

Introduction

Citrus Huanglongbing (HLB) is one of the most destructive globally distributed citrus diseases and is responsible for tremendous economic losses to citrus industries worldwide (Wang, 2019). CLas is the most widely distributed and is vectored by the Asian citrus psyllid, Diaphorina citri. Disease symptoms include blotchy mottle, yellow shoots, zinc deficiency, stunting, and twig dieback of citrus plants, as well as the dramatic decrease of fibrous root mass (Johnson et al, 2014; Wu et al, 2018). In addition to natural transmission by D. citri, CLas can be transmitted by graft inoculation within the same host genus and via at least three species of dodder (Cuscuta campestris, Cuscuta pentagona, and Cuscuta indecora) from citrus to nonnatural host plants, including periwinkle (Catharanthus roseus) (Garnier and Bové, 1983; Hartung et al, 2010), tomato (Solanum lycopersicum) (Duan et al, 2008), and tobacco (Nicotiana xanthi and Nicotiana benthamiana) (Francischini et al, 2007; Pitino et al, 2018). Periwinkle has been used as a surrogate for studying phylogenetic and taxonomic characteristics (Teixeira et al, 2008), distribution patterns (Ya et al, 2018), new detection method evaluation (Ding et al, 2017; Gottwald et al, 2020), genomic DNA enrichment and pathogenic effector screening of CLas (Zhang et al, 2011b; Jain et al, 2015), and therapeutic compound/molecule screening for HLB control (Zhang et al, 2010; Zhang et al, 2011a)

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