Global analysis of the AP2/ERF gene family in rose (Rosa chinensis) genome unveils the role of RcERF099 in Botrytis resistance

Abstract

Background: The AP2/ERFs belong to a large family of transcription factors in plants. The AP2/ERF gene family has been identified as a key player involved in both biotic and abiotic stress responses in plants, however, no comprehensive study has yet been carried out on the AP2/ERF gene family in rose (Rosa sp.), the most important ornamental crop worldwide.

Results: The present study comprises a genome-wide analysis of the AP2/ERF family genes (RcERFs) in the rose, involving their identification, gene structure, phylogenetic relationship, chromosome localization, collinearity analysis, as well as their expression patterns. Throughout the phylogenetic analysis, a total of 131 AP2/ERF genes in the rose genome were divided into 5 subgroups. The RcERFs are distributed over all the seven chromosomes of the rose, and genome duplication may have played a key role in their duplication. Furthermore, Ka/Ks analysis indicated that the duplicated RcERF genes often undergo purification selection with limited functional differentiation. Gene expression analysis revealed that 23 RcERFs were induced by infection of the necrotrophic fungal pathogen Botrytis cinerea. Presumably, these RcERFs are candidate genes which can react to the rose's resistance against Botrytis cinerea infection. By using virus-induced gene silencing, we confirmed that RcERF099 is an important regulator involved in the B.cinerea resistance in the rose petal.

Conclusion: Overall, our results conclude the necessity for further study of the AP2/ERF gene family in rose, and promote their potential application in improving the rose when subjected to biological stress.

Keywords: AP2/ERF gene family; Botrytis cinerea; Rosa sp.; Virus-induced gene silencing.

Fig. 1

Chromosome localization of rose AP2/ERF family members. The physical distribution of each RcERF gene is listed on the seven chromosomes of Rose chinensis

Fig. 2

Microsyntenic analyses of the rose AP2/ERF transcription factors in the Rose chinensis genome. Circular visualization of rose AP2/ERF transcription factors is mapped onto different chromosomes using Circos. The red lines indicate rose AP2/ERF genes having a syntenic relationship. The grey lines represent all syntenic blocks in the genome of R. chinensis

Fig. 3

Phylogenetic and gene structural analysis of rose AP2/ERF transcription factors. The phylogenetic tree is constructed by MEGA6.0 using a Neighbor-joining method. Numbers on the nodes of the branches represent bootstrap values. The gene structure diagram represents UTRs, exons and introns with green boxes, yellow boxes and gray lines, respectively. The scale at the bottom estimated the size of UTRs, exons and introns

Fig. 4

Phylogenetic analyses of the rose AP2/ERF transcription factors with disease-resistance-related AP2/ERF transcription factors from other plant species. The composite phylogenetic tree that included all rose AP2/ERF transcription factors and disease-resistance-related AP2/ERF transcription factors (in bold) from Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), soybean (Glycine max) and tomato (Solanum lycopersicum) were constructed by MEGA 6.0 with the neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. The bootstrap values are indicated on the nodes of the branches

Fig. 5

Validation of RNA-Seq results using qPCR. RcUBI2 was used as a housekeeping gene. Expression profile data of six RcERF genes at 30 hpi and 48 hpi after B. cinerea inoculation were obtained using qPCR. Error bar represent SD in three technical replicates. The primers used are listed in Supplementary Table S1

Fig. 6

Functional analysis of rose AP2/ERF transcription factor gene RcERF099. a Compromised B. cinerea resistance upon silencing of RcERF099 (TRV- RcERF099) was observed at 60 hpi post-inoculation. b. Quantification of B. cinerea disease lesions on TRV-RcERF099- and TRV-00-inoculated rose petal discs. The graph indicates the lesion size of three biological replicates (n = 48) with the standard deviation. c. Expression of RcERF099 relative to that during the control at 6 days of post-silencing. All statistical analyses were performed using Student’s t-test; ** p < 0.01