Identification of circular RNAs in resistant tomato genotype inresponse to ToLCBaV infection

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Tomato is a globally important vegetable crop and its cultivation is severly hampered by various pests and diseases including Tomato leaf curl virus (ToLCV). The losses due to infestation of ToLCV often exceeds 90 per cent (Varma and Malathi, 2003; Singh et al., 2015). Majority of genomic studies relied on molecular markers and functional analysis of genes. In order to understand the structural and functional concept of genomic regions, it is important to employ high- throughput next generation sequencing technologies (Barone et al., 2008; Wang et al., 2018). RNA sequencing also known as transcriptomics is one such technology that allows researchers to examine both known and unknown transcripts. Non-coding RNAs (ncRNAs) are transcripts that are not part of proteincoding genes (Wang et al., 2018). Among them Circular RNAs (CircRNA) are diverse and unique family of endogenous non-coding RNAs found in plant cells (Wang et al., 2018).

CircRNAs are abundant in the eukaryotic transcriptome. Their discovery and functional involvement in biological processes has opened up a new perspective so as to know how genomic regions interact in a variety of ways. However, their specific role is yet to be understood (Zhang et al., 2020; Litholdo et al., 2018). The majority of CircRNAs are conserved across species, although their expression varies according to tissue or developmental stage, as well as during biotic and abiotic stresses. CircRNAs interact with the transcriptional complex and influence the transcriptional and post-transcriptional regulation of gene expression (Zhang et al., 2020; Shao et al., 2021). They regulate parent gene expression by acting as miRNA sponges and RNA binding proteins (RBP) sponges (Hansen et al., 2013; Ashwal-Fluss et al., 2014; Shao et al., 2021). Their biogenesis competes with linear mRNA splicing to target alternative splicing mechanism of gene regulation (Shao et al., 2021). CircRNAs also regulate the translation of parental genes through interaction with trans-acting elements (Shao et al., 2021).

CircRNA were found to be differentially expressed during pathogen interaction in Arabidopsis (Sun et al., 2016; Zhang et al., 2020), pathogen invasion in kiwi fruit (Wang et al., 2017) and interaction with leaf curl virus in tomato (Wang et al., 2018); they also have regulatory roles in response to cotton verticillium wilt and maize Iranian mosaic virus (Xiang et al., 2018; Ghorbani et al., 2018). However, there is no information on the involvement of CircRNA in ToLCV tolerance in tomatoes. Keeping this in view, the present study investigated the potential role of CircRNA in regulating ToLCV resistance in tomato. Using high- throughput sequencing technology and appropriate bioinformatic tools, we analysed transcriptome data and identified CircRNAs. Abundance of CircRNAs, chromosome distribution and their corresponding genes were analysed and further the differential expression of few selected CircRNAs and their corresponding parent genes at different interval after ToLCBaV infection were analysed through gene expression studies.

CircRNAs are covalently closed non-coding RNA molecules. They predominantly comprising of exonic sequences and are spliced at canonical splice sites and were first discovered in humans and mouse, although they are found in all eukaryotes (Salzman, 2016). Use of highthroughput sequencing technologies and in silico analyses, have reported the CircRNAs-mediated gene regulation in plant immune system (Litholdo et al., 2018). In tomato plants, CircRNAs identification have been performed on tomato fruit ripening (Yin et al. 2018), tomato fruit coloration (Hong et al., 2020), fruit pigment accumulation (Yang et al., 2020), responsive to Phytophthora infestans (Zhou et al., 2020), TYLCV infection and tomato leaves responding to multiple stresses of drought and heat (Tan et al., 2017) and also low temperature treatments (Yang et al. 2020). In this study, identification of CircRNAs in tomato genotype resistant to ToLCBaV was examined and their functions in response to virus infection process are discussed.

In our study, a total 121 CircRNAs were generated from diverse genomic regions across all chromosomes in response to viral infection. Our result showed that Chr 1, 2, 4 and 6, had a greater number of induced CircRNAs (Fig. 1B). Few of them were involved in defence response (Table 2). Similar results were found where chromosome 01 had the most CircRNAs from susceptible tomato in response to TYLCV (Wang et al. 2018) and in response to multiple stresses of drought and heat (Zhou et al., 2020). Chr. 4 and Chr. 6 harbour TYLCV resistance loci Ty-5 (Hutton et al., 2012) and Ty-1/3 respectively (Dong et al., 2016) and induced CircRNAs on Chr. 4 and 6 might be having the regulatory roles on these resistance genomic regions. CircRNAs are mainly located at exons of genes, but scarcely distributed at introns or intergenic regions (Zuo et al. 2016; Yang et al. 2020). Similar pattern was observed in our experiment that CircRNAs were generated from various genomic regions and out of 193 total CircRNAs identified from both RC and RI, 103 (53 %) were from exonic region (Fig 1C). Similar trend was observed when the CircRNAs (62 % from exonic region) were analysed in susceptible tomato (Wang et al. 2018).

The expressions of exonic CircRNAs were significantly correlated with the expressions of parent genes (Ye et al., 2015; Wang et al., 2018). In our study, we observed a significant positive correlation between relative expression of selected CircRNAs and their parent genes (correlation coefficient r=0.89, 0.61 and 0.97 for HSP 0, POX, and SOD respectively) (Fig. 2). HSP90 function through 26S proteasome mediated proteolytic machinery in eukaryotic cells (Sadanandom et al., 2012). Due to decrease in the degradation of the TYLCV protein V2 by the 26S proteasome, silencing of Hsp90 led to enhanced accumulation of TYLCV CP and DNA levels as infection develops (Moshe et al., 2016). There is a significant positive correlation between 7:67566489|67566691 CircRNAs and its parent gene Solyc07g065840.2.1 (HSP90) (Fig. 2). TYLCV infection enhances defence mechanism through the activity of the antioxidant’s enzymes, i.e., SOD, CAT, PPO and POX in tomato (Dieng et al., 2011; Sofy et al., 2017). The corresponding biochemical activity of POX and SOD was in similar trend with the CircRNA expression across different intervals after ToLCBaV infection (Fig. 2 and 3).

CircRNAs in plants are differentially expressed both spatially and temporally in plants, acting as important functional modulators involved in biological processes (Pan et al., 2018; Wang et al., 2016; Zhou et al., 2017). CircRNAs (Slcirc017 parent gene) regulated TYLCV infection in susceptible plant and the silencing of its parent gene (Solyc01g080200.2) resulted in decreased TYLCV virus accumulation (Wang et al., 2018). In this study also differential expression of CircRNA parent genes (Solyc02g088950.2 and Solyc02g080530.3) was observed between control and infected conditions at different intervals (Fig. 2).

Fig. 3:
Response of SOD and POX activity in IIHR-2611 to ToLCBaV infection. Bar charts of different colour and with different letters are significantly different (P < 0.05, F = 1.702, df = 18, P = 0.021 for POX and F = 3.75, df = 18, P = 0.021) based on Tukey’s HSD test. Data presented as means ± SE (n = 3)

The results indicate that the CircRNAs- 2:51520741| 51530067 and 2:45295638|45295796 positively influence the plant response to disease through genes involved in ROS scavenging enzymes (SOD and POX) providing some insights into the role of CircRNAs in association with antioxidant enzymes against ToLCBaV. The increasing activity of peroxidase after virus infection (Fig. 2 and 3) might be due to structural defence of peroxidase which was known to perform polymerization, suberization, cell wall elongation, controlling virus multiplication and wounding (Bahar et al., 2020). TYLCV infection enhances the activity SOD and POX and further these enzymes activate the plant defence mechanisms (Dieng et al., 2011; Sofy et al., 2017).

CircRNAs analysed in this study might be acting as miRNA sponges and functioning through miRNA involved regulatory pathways or any of the other transcriptional, translational and posttranslational regulation mechanisms mentioned above. Furthermore, the precise mechanism by which CircRNAs regulate parent gene expression need to be investigated by identification of miRNA targets for the parent genes, in order to determine whether CircRNAs acting via miRNA mediated pathway or CircRNAs directly acting as parent gene regulator at transcription and translational level. The association between CircRNAs and interacting miRNAs was induced using the rice transgenic plants developed using agroinfection of rice calli with CircRNAs expression cassette (Sharma et al., 2021). There are various methods like artificial miRNA-mediated CircRNA knockdown, gain-offunction study, full-length CircRNA identification followed by CircRNA-protein interaction (Feng and Yu, 2021) to study and characterize the biological function of identified CircRNAs.

CircRNAs are emerging as a key player in RNA mediated gene regulation, having roles in several biological processes at both transcriptional and posttranscriptional stages. Many new studies on CircRNA profiling to diverse stresses found that the exonic CircRNAs positively regulates the expressions of their parent genes. This is the first report on CircRNAs for ToLCBaV resistance and we found a positive correlation between few CircRNAs and their parent genes. We hypothesised that these circRNAs must be acting as miRNA sponges and as regulators of miRNA mediated pathways in positively regulating their parental genes or acting as regulatory check points at transcription and translational level of parent gene expression. CircRNAs mediated regulation of some of their parent genes were found to be involved in host defence against viral disease. Further, research on understanding precise mechanism of these CircRNAs during viral infection and resistance against virus would help to identify target specific CircRNAs in plant viral disease resistance.

BC is thankful for PhD fellowship from the DBT - JRF Programme. KVR, ATS and KRM are thankful for the financial support received from DBT project titled “Introgression of Bogomovirus resistance genes in Tomato (Solanum lycopersicus L.) using MAS and genomic approach”.