Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease

Rawat et al. BMC Plant Biology (2015) 15:184 DOI 10.1186/s12870-015-0568-4 RESEARCH ARTICLE Open Access Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease Nidhi Rawat1, Sandhya P. Kiran2, Dongliang Du3, Fred G. Gmitter Jr3 and Zhanao Deng1* Abstract Background: Huanglongbing (HLB), the most devastating disease of citrus, is associated with infection by Candidatus Liberibacter asiaticus (CaLas) and is vectored by the Asian citrus psyllid (ACP). Recently, the molecular basis of citrus–HLB interactions has been examined using transcriptome analyses, and these analyses have identified many probe sets and pathways modulated by CaLas infection among different citrus cultivars. However, lack of consistency among reported findings indicates that an integrative approach is needed. This study was designed to identify the candidate probe sets in citrus–HLB interactions using meta-analysis and gene co-expression network modelling. Results: Twenty-two publically available transcriptome studies on citrus–HLB interactions, comprising 18 susceptible (S) datasets and four resistant (R) datasets, were investigated using Limma and RankProd methods of meta-analysis. A combined list of 7,412 differentially expressed probe sets was generated using a Teradata in-house Structured Query Language (SQL) script. We identified the 65 most common probe sets modulated in HLB disease among different tissues from the S and R datasets. Gene ontology analysis of these probe sets suggested that carbohydrate metabolism, nutrient transport, and biotic stress were the core pathways that were modulated in citrus by CaLas infection and HLB development. We also identified R-specific probe sets, which encoded leucine-rich repeat proteins, chitinase, constitutive disease resistance (CDR), miraculins, and lectins. Weighted gene co-expression network analysis (WGCNA) was conducted on 3,499 probe sets, and 21 modules with major hub probe sets were identified. Further, a miRNA nested network was created to examine gene regulation of the 3,499 target probe sets. Results suggest that csi-miR167 and csi-miR396 could affect ion transporters and defence response pathways, respectively. Conclusion: Most of the potential candidate hub probe sets were co-expressed with gibberellin pathway (GA)-related probe sets, implying the role of GA signalling in HLB resistance. Our findings contribute to the integration of existing citrus–HLB transcriptome data that will help to elucidate the holistic picture of the citrus–HLB interaction. The citrus probe sets identified in this analysis signify a robust set of HLB-responsive candidates that are useful for further validation. Keywords: Citrus, Gene co-expression analysis, Gene ontology, Huanglongbing, HLB resistance, Meta-analysis, miRNA network analysis, Common and R-specific probe sets * Correspondence: 1 University of Florida, Institute of Food and Agricultural Sciences, Gulf Coast Research and Education Center, Wimauma, FL 33598, USA Full list of author information is available at the end of the article © 2015 Rawat et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rawat et al. BMC Plant Biology (2015) 15:184 Background Citrus are among the most popular fruit crops in the world, providing energy (carbohydrates) and nutrients, and are an important component of the daily diet in many parts of the world [35]. Fresh citrus is also a good source of dietary fibre and vitamins B and C. Citrus fruits and fruit products are globally important from nutritional and economic perspectives [56]. However, there are various biotic and abiotic challenges to citrus production, among which Huanglongbing (HLB), or citrus greening disease, is the most devastating [5, 25, 61]. HLB was first reported in China in the early 1900s and is now well established in many citrus-producing regions, including India, China, the United States, Indonesia, the Philippines, the Arabian Peninsula, Brazil, and Africa [24, 25]. Brazil and the United States produce more than 90 % of the world’s supply of orange juice, and HLB is a current threat to the U.S. and the Brazilian citrus industry. HLB was first found in 2005 in Florida, the second-largest orange producing region in the world. Since then, HLB has reached epidemic proportions in Florida and has caused more than $4 billion in economic losses between 2005 and 2011 [27]. HLB attacks all important commercial citrus, including oranges, grapefruit, and tangerines [5, 20]. Sweet oranges and mandarins are considered highly susceptible, and sour oranges and grapefruits are moderately susceptible. It seems that some lemons are tolerant to HLB and some trifoliate orange (a close relative of citrus) are resistant of HLB disease [20]. HLB is a bacterial disease caused by gram negative and phloem-restricted Liberibacter species, which are vectored by the Asian citrus psyllid (Diaphorina citri Kuwayama) [21, 30]. D. citri feeds on new leaf growth, rendering twisted and curled leaves [36]. The disease can also be transmitted to healthy trees by grafting of diseased budwood [36]. Among the three known liberibacters that cause HLB disease, Candidatus Liberibacter asiaticus (CaLas) is the most widespread. The other two species are more geographically constrained; Ca. L. africanus is present primarily in Africa [30], and Ca. L. americanus has only been found in Brazil and China [54]. The bacterium resides in phloem tissues and causes phloem collapse, which leads to decreased productivity. The HLB disease causes a rapid tree decline with blotchy mottling of leaves, and small, misshapen, irregularly coloured, bitter fruit with aborted seeds [11, 23]. At present, there are no effective control methods for HLB, except for the use of HLBfree budwood for plant propagation, removal of infected trees to minimize inoculum, and insect vector control [5]. The full genome sequencing of CaLas (1.23 Mb) has made genome-based identification of virulence factors associated with HLB symptoms possible [15]. However, CaLas has not been cultured and a full understanding of the molecular basis of citrus–HLB interactions is lacking [17]. It Page 2 of 21 is difficult to create HLB-tolerant citrus cultivars via conventional breeding because of a lack of any known resistance (R) genes against HLB and because of the complex biology of the citrus host. However, examination of citrus genomics through transcriptome and proteome analysis can elucidate the differentially expressed genes/proteins as pot (...truncated)