Transcriptional and post-transcriptional regulation of RNAi-related gene expression during plant-virus interactions

As sessile organisms, plants encounter diverse invasions from pathogens including viruses. To survive and thrive, plants have evolved multilayered defense mechanisms to combat virus infection. RNAi, also known as RNA silencing, is an across-kingdom innate immunity and gene regulatory machinery. Molecular framework and crucial roles of RNAi in antiviral defense have been well-characterized. However, it is largely unknown that how RNAi is transcriptionally regulated to initiate, maintain and enhance cellular silencing under normal or stress conditions. Recently, insights into the transcriptional and post-transcriptional regulation of RNAi-related genes in different physiological processes have been emerging. In this review, we integrate these new findings to provide updated views on how plants modulate RNAi machinery at the (post-) transcriptional level to respond to virus infection.


Introduction
Plants are persistently challenged by various phytopathogens. Among them, viruses, as obligatory intracellular parasites, can cause severe diseases and viral epidemics on all major crops of agronomic importance. To protect themselves, plants have evolved multilayered defense mechanisms against viruses including physical barriers, innate immunity, RNAi, and autophagy (Haxim et al., 2017;Ismayil et al., 2020;Lopez-Gomollon & Baulcombe, 2022;Soosaar et al., 2005).

Open Access
Stress Biology † Qian Gong, Yunjing Wang and Zhenhui Jin contributed equally to this work. *Correspondence: yuleliu@mail.tsinghua.edu.cn a sequence homology-dependent mechanism (Peters & Meister, 2007;Vaucheret, 2008). The association of RISC with complementary target RNAs leads to cleavage, degradation, or translational inhibition of the cognate viral RNAs (Fang & Qi, 2016;Garcia-Ruiz et al., 2015;Jaubert et al., 2011;Wu et al., 2015;Zhang et al., 2006), while the interaction with target viral DNA causes modification of DNA and/or histones, result in transcriptional repression (Raja et al., 2008) (Fig. 1). Although the functions of genes encoding proteins involved in RNA silencing were well-characterized in plants, the regulatory mechanism of their transcription remains elusive. In this review, we will highlight recent advances on transcriptional and post-transcriptional regulation of RNAi-related gene expression and discuss how miRNAs, phytohormones, and viral pathogens influence RNAi-related gene expression during the plant-virus warfare.

Regulation of miRNAs and their roles in plant-virus interactions
Most plants possess a large number of microRNA (MIR) genes, mainly in intergenic regions throughout the genome . Most MIR genes possess their own transcriptional unit. MIR genes are transcribed into pri-miRNAs by RNA polymerase II (Pol II) (Stepien et al., 2017;Xie et al., 2005), and pri-miRNAs are ultimately processed into small (size of 20-24 nucleotides long) ssRNAs, termed as miRNAs (Liu et al., 2012;Song et al., 2010;Zhu et al., 2013). The mature miRNAs guide strand can be loaded into an AGO protein to form RISC. The PIWI domain of AGO proteins forms an RNase H-like fold with a slicer endonuclease activity, which is capable of cleaving target RNAs that are complementary to the loaded guide strand (Song et al., 2004). MiRNAs play key roles in the regulation of their targeted genes expression in plants.
In plant-virus interactions, miRNAs were used as weapons for both host and pathogen. MiRNAs play important roles in antiviral immunity by targeting endogenous genes, including RNA silencing components, hormone signaling pathways, and nucleotide binding site-leucine-rich repeat (NBS-LRR) resistance (R) genes (Table 1) (Jin et al., 2021;Mlotshwa et al., 2008;Zhang et al., 2016). For example, upon Rice stripe virus (RSV) infection, miR528 becomes preferentially associated with AGO18, leading to increased L-ascorbate oxidase (AO) activity, increased basal active oxygen accumulation and enhanced antiviral defense in rice . RSV infection also induce miR444 transcription and diminishes the repressive effects of the MADS box genes on RDR1 transcription, thus activating RDR1 dependent antiviral silencing pathway . In terms of miRNAs regulation of R genes, bra-miR1885 which targets TIR-NBS-LRR class R gene is specifically induced by TuMV infection in brassica (He et al., 2008). MiR482/2118 family is found to target NB-LRR encoding genes in tomato, while nta-miR6019 and nta-miR6020 guide cleavage of transcripts of tobacco NB-LRR immune receptor N that confers resistance to Tobacco mosaic virus (TMV) (Li et al., 2012a;Shivaprasad et al., 2012;Zhai et al., 2011). In addition, RNAi can be controlled by miRNAs` feedback regulation of RNAi related genes expression. For example, miR162, miR168, and miR403 targets DCL1, AGO1, and AGO2/3 mRNA, respectively (Allen et al., 2005;Vaucheret et al., 2004;Xie et al., 2003).
Although alternation of miRNA expression or activity during viral infection has been found extensively, the regulation mechanism for these cases is largely unknown. Both plant and viral protein can cause differential miRNA expression and activity. For examples, rice SQUAMOSA Promoter Binding Protein-Like 9 (SPL9) binds to miR528 promoter and activates miR528 gene expression as the transcription factor (TF) in rice plants (Yao et al., 2019). In addition, RSV infection enhances jasmonic acid (JA) biosynthesis and signaling of the infected plants, leading to the release of JA-induced TF JAMYB. JAMYB binds to and activates the AGO18 promoter. AGO18 is found recruiting a large amount of miR168 through small RNA deep sequencing analyses of purified AGO18-containing complexes, further relieves the repression of miR168 on AGO1 mRNA in RSV-infected rice (Wu et al., 2015;Yang et al., 2020). As a major effector of antiviral RNA silencing, AGO1 associates with vsiRNAs and mediates degradation of viral RNAs (Wu et al., 2015;Yang et al., 2020). Little is known about how plants sense initial cues to mobilize RNAi. Recently, we found that mechanical wounding or aphid feeding to Nicotiana benthamiana cells during virus intrusion activates calmodulin-binding transcription activator-3 (CAMTA3) function, which directly binds to Bifunctional nuclease-2 (BN2) and RDR6 promoters and induces their transcription. BN2 stabilizes AGO1/2 and DCL1 mRNA levels by degrading their cognate microRNAs . Therefore, multiple RNAi components are primed for combating virus invasion. Viruses also took full advantage of miR-NAs for the effective infection. RSV NS3 (P3) Protein suppresses RNA silencing to regulate the expression of multiple host resistance-associated miRNAs upon RSV infection (Shen et al., 2010;Zheng et al., 2017). Besides, many viruses encode viral suppressor of RNA silencing (VSR) such as P19 from Tombusvirus and P1/HC-Pro from TuMV to enhance virus infection by regulating host miRNAs biogenesis, activity, or accumulation Zhang et al., 2006).

Regulation of ta-siRNAs and their roles in plant-virus interactions
Another class of endogenous sRNAs which have important roles during plant-virus interactions are trans-acting siRNA (ta-siRNA). Ta-siRNA is a subset of phasiRNAs encoded by TAS genes that can regulate target genes via mRNA cleavage in trans (Allen et al., 2005;Fei et al., 2013;Yoshikawa et al., 2005). The biogenesis of ta-siR-NAs is initiated by miRNA-mediated cleavage of TAS transcripts. The cleaved RNAs are copied into dsRNAs by RDR6, and dsRNAs are cleaved to generate multiple ta-siRNAs by type III ribonuclease in a phased manner (Axtell et al., 2006). To date, four families of TAS genes with eight loci have been discovered in the Arabidopsis genome: TAS1, TAS2, TAS3, and TAS4 (Chen, 2009). TAS1 and TAS2 require miR173 for ta-siRNA biogenesis, whereas TAS3 and TAS4 require miR390 and miR828, respectively (Allen et al., 2005;Peragine et al., 2004;Rajagopalan et al., 2006;Vazquez et al., 2004;Yoshikawa et al., 2005). TAS1 and TAS2 only exist in certain plant species, however, TAS3 and TAS4 are conserved (Allen & Howell, 2010; Xia et al., 2017).
Ta-siRNAs are involved in plant-virus interactions and induced during the infection of plant with pathogens including ToLCNDV (Singh et al., 2015). In addition, ta-siRNAs are generated and transported systemically within 4 to 6 h of primary pathogen infection to induce systemic acquired resistance (SAR) (Shine et al., 2022). Viruses also employ different strategies to suppress ta-siRNAs generation. For instance, a small peptide VISP1 is reported to compromise antiviral immunity by inducing autophagic degradation of SGS3 to inhibit SGS3/RDR6dependent viral siRNA amplification and endogenous ta-siRNAs biogenesis during CMV infection (Tong et al., 2021). CMV 2b protein also interferes with the production of ta-siRNAs through interaction with AGO1 (Feng et al., 2013). Apart from CMV, transactivator/viroplasmin (TAV) protein of Cauliflower mosaic virus (CaMV), p2 protein of RSV, TGBp1 of Plantago asiatica mosaic Gong et al. Stress Biology (2022) 2:33 virus (PlAMV), and coat protein (CP) of HCRSV are capable of interfering with ta-siRNAs biogenesis, mainly via interaction with SGS3/RDR6 bodies (Du et al., 2011b;Meng et al., 2008;Okano et al., 2014;Shivaprasad et al., 2008). In addition, syn-tasiRNAs can be designed to target virus in plants. Syn-tasiRNA contains a functional TAS precursor in which a subset of the endogenous ta-siRNA sequences is substituted by one or several designed syn-tasiRNA sequences in tandem Cisneros & Carbonell, 2020;Miao et al., 2021). Indeed, syn-tasiRNAs can confer virus resistance in multiple plant species (Carbonell & Daros, 2017;Carbonell et al., 2019aCarbonell et al., , 2019b.

Effects of phytohormone on the expression of RNAi components
Phytohormones are required for plant development and response to biotic or abiotic stresses. Numerous findings have revealed the significance of not only individual phytohormones or separate signaling cascades but also complex network of intersecting hormone signal pathways in antiviral immunity (Alazem & Lin, 2015;. However, the cross-talk between phytohormones and RNAi is very complicated, and more attention is needed to understand the effects of hormones on the regulation of antiviral RNAi (Fig. 2).
JA is a key regulator of defense responses to necrotrophic pathogens as well as insect infestation in plants (Chini et al., 2016;Wasternack, 2014;Yan et al., 2018;Zhang et al., 2017). JA is also involved in plant antiviral defense (Jia et al., 2016;Yang et al., 2020). However, the connection between JA and RNAi pathway has not been clear. A recent report has linked JA to the expression of an RNAi component during virus infection. In this study, RSV CP triggers JA biosynthesis and signaling, leading to the degradation of JAZ proteins and the release of TF JAMYB. JAMYB binds to and activates AGO18 promoter to enhance the transcription of AGO18, which further increases rice antiviral RNAi defense by sequestering miR168 and releasing AGO1 mRNA (Wu et al., 2015;Yang et al., 2020) (Fig. 2).
Salicylic acid (SA) plays a vital role in plant immunity including antiviral defense (Yan & Dong, 2014). SA treatment significantly induces transcription of DCL1/2, RDR1/2 in tomato, leading to a repression of Tomato mosaic virus (ToMV) infection (Campos et al., 2014). Similarly, exogenous SA application significantly triggers the transcription of RNAi pathway genes including DCL1/2/4, RDR2/3a, RDR6a, and AGO1/4 in tomato, enhances the resistance to Tomato yellow leaf curl virus (TYLCV) (Li et al., 2018). Besides, SA is able to induce RDR1 expression in tobacco and Arabidopsis to defense  Rakhshandehroo et al., 2017). The biocontrol agent ZhiNengCong (ZNC), which is the extraction of an endophytic fungus, increases SA content along with positive regulation of DCL3, AGO10, and other RNAirelated gene expression to enhance tobacco resistance against PVX in wild-type tobacco plants, but failed to induce those protective effects in transgenic NahG plants expressing SA-degrading enzyme salicylate hydroxylase (Peng et al., 2020). Interestingly, some SA-related TFs are co-expressed with AGO, DCL, and RDR genes, and the promoter regions of these AGO, DCL, and RDR genes arepredicted to contain the multiple binding sites for the corresponding SA-related TFs (Alazem et al., 2019) (Fig. 2). These results indicate that SA is able to modulate RNAi-related gene expression to repress virus infection. SA and abscisic acid (ABA) are often antagonistic and regulate different stress responses, however, they have similar effects on antiviral immunity, which are partially achieved through RNAi pathway. The regulatory role of ABA in RNAi pathway isuncovered by Arabidopsis ABA deficient mutants aba1-5 (Leon-Kloosterziel et al., 1996). In aba1-5 plants, the expression level of AGO1 is significantly increased, suggesting that ABA negatively regulates AGO1 expression (Li et al., 2012b). Additionally, miR168a, a negative regulator of AGO1, is upregulated by ABA (Laubinger et al., 2010;Li et al., 2012b). Apart from that, impairment of the ABA pathway in Arabidopsis thaliana reduces the accumulation of AGO2 and weakens resistance to PVX (Jaubert et al., 2011). ABA upregulates the expression of AGO2, AGO3, and AGO4 to enhance resistance to Bamboo mosaic virus (BaMV) (Alazem et al., 2017). ABA also positively regulates RDR6 gene expression and post-transcriptional gene silencing in rice cells (Yang et al., 2008) (Fig. 2). Notably, multiple RNA-silencing mutants, such as dcl1, ago1, hen1, se-1, and hyl1 have ABA-hypersensitivity (Li et al., 2012b;Lu & Fedoroff, 2000;Zhang et al., 2008). These studies have allured more attention to the effects of hormones on transcriptional regulation of antiviral RNAi components. In view of the cross-talk between phytohormones and RNAi, there are still some crucial unsolved questions that need to be further characterized. For instance, 1) we need further investigation into how these hormones affect the key genes (DCLs, RDRs, and AGOs) in RNAi pathway. 2) Apart from DCL, RDR, and AGO family genes, are there any other genes which regulate or maintain the integrity of RNAi pathway modulated by these hormones? 3) Some genes are able to be transcriptionally regulated by multiple hormones with antagonism pattern, therefore, how hormones coordinate the regulation of RNAi-related gene expression? For instance, SA and ABA exhibited mutual antagonism of AGO1 and RDRs expression (Alazem et al., 2019). ABA clearly induced expression of those genes only in the SA mutant sid2-1, however, both SA and ABA show similar regulation for other genes, for example, ABA-mediated AGO2 induction is SA-dependent (Alazem et al., 2019). Besides, although the contribution of SA/JA signaling molecules in plant defense differs and depends on the type of invading pathogen, these two signaling pathways influence each other via a complex network of synergistic and antagonistic interactions (Alazem & Lin, 2015;. The RNAi regulation by phytohormones are not simple linear or isolated cascades, but exhibit cross-talk with each other. Alteration in endogenous phytohormone levels seems to be a direct consequence of virus infection and is tightly coordinated with viral movement, replication, symptom development, and defense responses (Casteel et al., 2015;Tao et al., 2017;Zhao & Li, 2021). Hijacking host components in the phytohormone pathways is a common strategy in viral pathogenesis (Zhao & Li, 2021). Identifying the roles of phytohormones in viral infection and cross-talk with antiviral RNAi defense among different phytohormones pathways are challenges for the forthcoming years. We still lack specific molecular basis of phytohormones regulation of RNAi-related gene transcription. The comprehensive mechanism of signal integration among multiple phytohormones to regulate RNAi also needs further investigation.

The effect of viral infection on RNAi-related gene expression
Viral infection often activates or up-regulates expression of host RNAi-related genes and this virus-resistant strategy seems more general in Solanaceae family plants (Fig. 3). For example, the transcription of multiple AGOs (AGO1, AGO2, AGO4, and AGO10), RDR6, DCL2, and DCL4 are upregulated with CMV, PVY, or TMV infection in pepper (Qin et al., 2018). Also, expression profiling of genes in TYLCV infected tomato showed that multiple RNAi core genes including SlDCL1/2/3, SlRDR2, SlRDR6, and five AGO genes (SlAGO1a, 1b, 4a, 4b, and 5) are triggered with high level expression in response to virus infection (Bai et al., 2012). In Nicotiana benthamiana, expression of repeat sequence fragments from both Pepper golden mosaic virus (PepGMV) and Tomato chino La Paz virus (ToChLPV) are able to upregulate DCL2/3/4, AGO1/2/3, AGO7, AGO10, and RDR6 transcripts (Vargas-Salinas et al., 2021). AGO1 mRNA level is elevated in Cymbidium ringspot virus (CymRSV) infected plants (Havelda et al., 2008). RDR1 is also reported to be virus or SA inducible in different plants including Arabidopsis, Nicotiana, Medicago truncatula, maize (Zea mays), and rice (Oryza sativa) (Alamillo et al., 2006;Du et al., 2011a;He et al., 2010;Satoh et al., 2010;Yang et al., 2004). For instance, expression of the Nicotiana tabacum RDR1 gene is induced by TMV, PVY, and PPV (Rakhshandehroo et al., 2009). In cucumber, four RDR1 homologous genes are regulated with different expression profiles during virus infection. RDR1b is constitutively expressed at a high level only in resistant plants, whereas RDR1c1 and RDR1c2 are barely expressed in healthy plants, but induced to high levels by RNA and DNA virus infection (Kumari et al., 2021;Leibman et al., 2018). Besides, RSV infection induces miR444 accumulation, which enhances OsRDR1 expression, leading to rice resistance to RSV infection . Although similar results have been repeatedly achieved, the regulation mechanism for those cases, especially the initial cue which provokes these responses is still elusive. Moreover, it also remains unclear how RNAi-related genes are transcriptionally and post-transcriptionally regulated. Recently, we revealed that a Ca 2+ -calmodulin-CAMTA3 cascade which may supply some missing part of the "puzzle". We found that mechanical wounding or aphid feeding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling . A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of CAMTA3, which activates RDR6 and BN2 transcription. BN2 stabilizes mRNAs encoding key RNAi machinery components AGO1/2 and DCL1 by degrading their cognate microRNAs . Consequently, multiple RNAi-related genes expression is primed for combating virus invasion. These findings demonstrate that calcium signaling can act as a cue to up-regulate and tune the RNAi machinery.
Furthermore, to survive, viruses encode proteins to counteract host RNAi-related gene activation as well (Fig. 4). For instance, tombusvirus infection enhances mRNA level of AGO1 to resist virus infection, however, tombusvirus p19, as a RNA-silencing suppressor, mediates the induction of the miR168 expression to down-regulate endogenous AGO1 mRNA level and inhibit the translational capacity of AGO1 mRNA (Varallyay et al., 2010). Another example is the CMV encoded 2b suppressor protein. CMV 2b is found to inhibit miRNA pathways by blocking AGO1 cleavage activity to upregulate miR168 and miR162 levels. Since miR162 and 168 negatively regulate the RNAi by targeting DCL1 and AGO1 mRNAs, respectively, CMV 2b attenuates antiviral RNAi and counters host defense (Zhang et al., 2006 , 2020), indicating that the upregulation of miR168 commonly occurs during plant-virus interactions, and it is not related to the host species and the mode in which different VSRs act . Moreover, Tomato yellow leaf curl China geminivirus (TYLCCNV) encodes VSR βC1 to fight against the host RNAi-mediated defense. βC1 induces a calmodulin-like (Nbrgs-CaM) gene expression, and Nbrgs-CaM suppresses the production of secondary siRNAs, likely through repressing RDR6 expression (Li et al., 2014). Another interesting strategy by which geminiviruses employ is uncovered recently. During virus invasion, the rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of CAMTA3, which activates RDR6 and BN2 transcription. BN2 stabilizes AGO1/2 and DCL1 mRNAs, by degrading their cognate microRNAs. V2 proteins encoded by Cotton Leaf Curl Multan virus (CLCuMuV) and TYLCCNV can disrupt the calmodulin-CAMTA3 interaction, which further impair CAMTA3-mediated transcriptional activation of both RDR6 and BN2 to suppress antiviral RNAi .

Conclusions and perspectives
Over the past decades, RNAi has become a research hotspot in the research field of plant-virus interactions. RNAi plays a significant role in regulating defense against virus invasion by degrading RNA or modifying DNA through siRNAs. However, how RNAi is transcriptionally regulated to initiate, maintain, and enhance cellular RNAi machinery during virus infections still await to be uncovered. Current studies are extending knowledge concerning the correlation between RNAi and different physiological factors such as phytohormone or pathogens, however, more information is required for elucidating the mechanism of fine-tuning RNAi machinery on RNAi-related gene transcriptional control. To unveil the truth, we need to confront following challenges in forthcoming years. 1) New genes or small RNAs associated with antiviral RNAi need to be identified. 2) How RNAi key genes are transcriptionally regulated by phytohormones or virus infection still awaits to be discovered. 3) Since transcriptional regulation of RNAi machinery is associated with multiple aspects, challenge lies in deciphering emerging picture of complex mechanisms which are not simple linear or isolated cascades, but exhibit cross-talk. Furthermore, challenge still lies in translating the basic knowledge gained from model species to crops. In summary, addressing how RNAi is transcriptionally and post-transcriptionally regulated in plantvirus interactions will advance our understanding of RNAi machinery and elucidate how plant recognizes different stress and responses through RNAi. Future research in this field will surely yield more exciting discoveries and support development of plant antiviral immunity.