Small RNAs: Efficient and miraculous effectors that play key roles in plant–microbe interactions

Abstract Plants' response to pathogens is highly complex and involves changes at different levels, such as activation or repression of a vast array of genes. Recently, many studies have demonstrated that many RNAs, especially small RNAs (sRNAs), are involved in genetic expression and reprogramming affecting plant–pathogen interactions. The sRNAs, including short interfering RNAs and microRNAs, are noncoding RNA with 18–30 nucleotides, and are recognized as key genetic and epigenetic regulators. In this review, we summarize the new findings about defence‐related sRNAs in the response to pathogens and our current understanding of their effects on plant–pathogen interactions. The main content of this review article includes the roles of sRNAs in plant–pathogen interactions, cross‐kingdom sRNA trafficking between host and pathogen, and the application of RNA‐based fungicides for plant disease control.

successful infection and causing disease (Hua et al., 2018;Thomma et al., 2011). Some plants have evolved a second type of immune response to inhibit pathogen invasion, called effector-triggered immunity (ETI). ETI acts largely inside the plant cell via polymorphic proteins containing a nucleotide-binding (NB) domain and a leucinerich repeat (LRR) structure, which are encoded by plant disease resistance (R) genes .
For the plant, the successful initiation of the innate immune response on pathogen infection requires comprehensive and accurate gene expression reprogramming and communication between the host and microorganisms. Recently, several investigations have shown that many small RNAs (sRNAs) are involved in genetic expression and reprogramming affecting plant-pathogen interactions (Huang et al., 2019). Plant sRNAs (18-30 nucleotides [nt] in length) can be classified into two major categories, termed microRNAs (miRNA) and small interfering RNAs (siRNA), according to their biogenetic pathways and morphology (Achkar et al., 2016;Cui et al., 2017;D'Ario et al., 2017). There are also further special classes, such as trans-acting small interfering RNAs (ta-siRNAs), small nuclear RNA (snRNA, also referred to as U-RNA), natural antisense small interfering RNAs (nat-siRNAs), long siRNAs (lsiRNAs), and small nucleolar RNA (snoRNA) Katiyar-Agarwal & Jin, 2010;Shahid et al., 2018). The miRNAs are generated from singlestranded RNAs (ssRNA) with imperfectly base-paired stem-loop structures; the siRNAs are generated from long double-stranded RNAs (dsRNAs) and by RNA-dependent RNA polymerase (RDR) activity (D'Ario et al., 2017;Devert et al., 2015;Islam et al., 2017;Katiyar-Agarwal & Jin, 2010). Recently, it was found that the DICER-LIKE PROTEIN 3 (DCL3) produces 24-nt siRNAs that determine the specificity of the RNA-directed DNA methylation pathway. The 24nt siRNA length dependence is critical for the separation between the 5′-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. The machinery for RNA interference (RNAi) consists of three core components: RDRs for biosynthesis of dsRNA from an ssRNA; DCL, for cleaving ssRNA with imperfectly base-paired stem-loop structures or dsRNA into sRNAs; and Argonaute (AGO) proteins, binding sRNAs to form RNAinduced silencing complexes (RISC) for leading the target mRNA to cleavage or translation suppression (Elbashir et al., 2001;Islam et al., 2018;Zhu et al., 2019). The mechanism of cross-kingdom RNAi has also been considered and studied in plant-pathogen interactions (Kulshrestha et al., 2020;Weiberg et al., 2013;Zotti et al., 2018).
Recently, studies have discovered that sRNAs function as pathogen effectors to regulate host immunity and pathogen infection by silencing target genes in the host Weiberg et al., 2014;Weiberg & Jin, 2015). However, a large number of scientific problems behind this mechanism still need to be studied and expounded.
In this review, we summarize the effects of sRNA on plantpathogen interactions and highlight the recent discoveries of crosskingdom sRNA trafficking between host and pathogen. Finally, we also discuss the possibility of using RNA-based fungicides for plant protection.

| THE ROLE OF sRNA s IN PL ANT-PATHOG EN INTER AC TI ON S
Various plant diseases caused by pathogens, including oomycetes, fungi, bacteria, viruses, nematodes, mycoplasma, viroids, and other parasites, have caused great damage to crop production and resulted in huge economic losses (Figure 1) (Islam et al., 2018). A number of plant endogenous sRNAs are involved in plantpathogen interactions and regulation of the immune responses. It has been demonstrated that sRNAs are involved in the plant defence response through different pathways that actively regulate plant immunity to pathogen infection by tackling PAMPs and effectors. The first miRNA found to be involved in plant immunity is the well-known miR393, which is induced by flg22 (a PAMP); it activates the PTI by silencing the auxin receptors to affect the auxin signalling pathway in Arabidopsis (Huang et al., 2019;Navarro et al., 2006). The first sRNA found to be involved in plant immunity was nat-siRNAATGB2, which is specifically and highly induced by Pseudomonas syringae pv. tomato (Pst) carrying the effector AvrRpt2; it promotes ETI by silencing a pentatricopeptide repeatlike protein (a negative regulator of plant defence) (Huang et al., 2019;Katiyar-Agarwal & Jin, 2010). Table 1 shows the sRNAs involved in plant-pathogen interactions and regulation of immune responses to a variety of pathogens.

| sRNAs in bacteria-plant interactions
Recently, many sRNAs that directly take part in the response to bacterial diseases have been identified (Table 1 and Figure 1). miR393b*/MEMB12 are important effectors or regulators in plant antibacterial immunity (Zhang, Zhao, et al., 2011). In Arabidopsis, miR393b* (the complementary strand of miR393) has been identified as an AGO2-bound sRNA, which could target MEMB12 encoding a SNARE protein localized in Golgi apparatus. Both miR393b* overexpression and memb12 mutation promoted the secretion of PR1 in Arabidopsis in response to Pst DC3000 infection (Zhang, Zhao, et al., 2011). A large number of studies have implicated miR393 as being strongly involved in ETI. It was also found that miR393 was significantly repressed, resulting in the target gene LecRLK (lectin receptor-like kinase) being upregulated to enhance perception ability of bacterial lipopolysaccharide in Arabidopsis (Djami-Tchatchou & Dubery, 2015). The overexpression and repression of miR393, respectively, suppressed and induced the expression of LecRLK in Arabidopsis treated with lipopolysaccharide (Djami-Tchatchou & Dubery, 2019). Repression of auxin signalling constitutes part of a plant's defence response to bacterial infection (Gao & Jin, 2011;Navarro et al., 2006;Pruss et al., 2008). It was observed that miR160 and miR167 were induced in response to Pst DC3000 hrcC − and flg22, and that miR160a-overexpressing plants increased callose deposition after treatment with Pst DC3000 hrcC − and flg22 (Yao et al., 2013). Moreover, investigation of the tumours caused by infection of Agrobacterium tumefaciens revealed that miR167 and miR393 were significantly down-regulated and that mutants of these miRNAs showed hypersusceptibility to the bacterium (Li et al., 2010). The miR398 production, targeting CSD1, CSD2 (copper superoxide dismutases) and COX5b-1 (a cytochrome coxidase subunit V), was reduced in plants challenged with avirulent strains such as Pst DC3000 avrRpm1 and Pst DC3000 avrRpt2 (Jagadeeswaran et al., 2009). It was also found that flg22 suppresses miR398b accumulation. In contrast, the expression of the miR398 target genes COX5b-1, CSD1, and CSD2 is increased (Li et al., 2010). Significantly suppressed miR398 was also observed in citrus plants infected with pathogenic bacteria of the genus "Candidatus Liberibacter" . Researchers have screened various other miRNA families that are involved in antibacterial defence in plants by deep-sequencing. For example, Zhang et al. described the expression of 20 diverse miRNA families on the invasion of Pst DC3000 in Arabidopsis; most of the target genes were involved in the synthesis and signalling pathways of various hormones such SA, jasmonic acid (JA), and abscisic acid (ABA) . The involvement of hormone pathways such as SA, JA, and ABA in host defence has been well studied. Thus, these studies show that the miRNAs normally facilitate the fine-tuning of defence responses rather than targeting some genes involved in the plant immune system directly (Ballaré, 2015;Berens et al., 2017;Ludwig-Müller, 2015;Qi et al., 2012;Ramegowda & Senthil-Kumar, 2015;Sanchez-Vallet et al., 2012;Song et al., 2013;Tamaoki et al., 2013).
Like miRNA, siRNAs have also been reported to be involved in the interaction between plants and bacteria, such as nat-siRNA, nat-siRNAATGB2, and some lsiRNAs (Islam et al., 2017;Weiberg & Jin, 2015). Five lsiRNAs were induced in plants against Pst avrRpt2 infection and an endogenous siRNA siRNAATGB2 was identified that is derived from the natural antisense transcripts pair ATGB2-PPRL, functioning for plant resistance to Pst avrRpt2 (Katiyar-Agarwal et al., 2006. The activation of secondary siRNA production and amplification of silencing signals is dependent on RDR6. An miR472-RDR6 silencing pathway has been reported that is required for enhancing plant defence against P. syringae. The miR482/2118 family suppresses NB-LRRs by production of short tandem target mimic RNAs, dependent on RDR6, to enhance plant resistance to P. syringae in tomato (Boccara et al., 2014;Canto-Pastor et al., 2019).  (Islam et al., 2017;Schmidtke et al., 2012Schmidtke et al., , 2013. Here, the involvement of sRNAs in bacteria-pathogen interactions is summarized. Although miRNAs or siRNAs are vital components of various defence-related pathways, the specificity function and contributions on targets of these sRNAs still need to be explored. GhmiR477-silencing decreased plant resistance to V. dahliae infection while the knockdown of GhCBP60A increased plant resistance to the pathogen . sRNA also plays an important role in the interaction between oomycetes and host plants.  Huang et al., 2017;Qiao et al., 2015). The WY domain in PSR1 is required for infection and RNA silencing suppression activity (Zhang, Jia, et al., 2019).

| sRNAs in fungus/oomycete-plant interactions
Deep sequencing of sRNA libraries from susceptible and resistant rice lines uncovered the important role that sRNAs play in enhancing immunity against rice blast disease caused by Magnaporthe oryzae (Li et al., 2014). miR160a and miR398b overexpression lines showed resistance to M. oryzae by increasing hydrogen peroxide accumulation and raising the expression of pathogenicity-related genes (PR genes) to decrease fungal growth in the rice plants (Li et al., 2014). It was also found that Osa-miR7695 overexpression resulted in plant resistance to M. oryzae and thus that Os-miR7695 modulated plant immunity, illustrating a novel regulatory network targeting natural resistance-associated macrophage protein 6 (OsNramp6) (Campo et al., 2013). OsDCL4 for biogenesis and OsAGO18 for function (Niu et al., 2017).
Other studies have found that 468 known mature miRNAs and 747 putative novel miRNAs may be involved in rice-R. solani interactions (Srikakulam et al., 2022  were greatly up-regulated in response to turnip mosaic virus infection, and the mechanism of bra-miR158 and bra-miR1885 regulating plant immunity by targeting TIR-NBS-LRR was clarified (Hewezi et al., 2008). In addition, the expression level of miR482 decreased, showed more susceptibility to the virus, whereas the opposite response was observed in the miR171b target mimic lines (Tong et al., 2017). Although many sRNAs have been reported to be involved in plant-virus interactions, the molecular mechanism of sRNAs response to viral infections is not yet known. It will therefore be interesting to investigate the role of RNA in viral infections and the pathways associated with the observed response in the future.

| sRNAs in virus-or viroid-plant interactions
Viroids are small (250-400 nt) single-stranded, circular RNA, pathogens, and infect several crop plants causing diseases of economic importance (Navarro et al., 2021). Viroids are known to initiate a range of sRNAs in plants. For example, RNA silencing is targeted and activated by potato spindle tuber viroid (PSTVd) in infected potato plants (Dalakouras et al., 2015).  (Wang et al., 2015). For example, several lncRNAs were differentially expressed during TYLCV infection; silencing of two of these, lncRNA-0761 and lncRNA-0049, resulted in an increase in disease severity (Wang et al., 2015). In another work on the identification of lncRNAs as key regulators of gene expression in the tomato-TYLCV system, RNA-sequencing revealed different patterns of lncRNAs and circular RNAs (circRNAs) from plants infected with TYLCV compared to healthy plants. Silencing of sly-lnc0957 resulted in enhanced resistance to TYLCV in susceptible tomato cultivars. In this case, the lncRNA was demonstrated to be a negative regulator of TYLCV infection (Wang, Yang, et al., 2018). Similarly, in response to maize Iranian mosaic virus infection, the maize plants showed different expressions of circRNAs; deep sequencing identified 155 circRNAs were up-regulated whereas five were down-regulated. Among these were 23 maize miRNAs that were responsible for regulating plant development and metabolism (Ghorbani et al., 2018). Moreover, cucumber green mottle mosaic virus infection of watermelon results in differential expression of 548 and 67 lncRNAs, which are responsible for phenylalanine metabolism, citrate cycle, and endocytosis (Shrestha & Józef, 2020;Sun et al., 2020). Taken together, all the above results demonstrate the complex nature of lncRNAs and cir-cRNAs in defence signalling pathways and indicate their function in the regulation of defence response genes. Therefore, studying the function of lncRNAs and circRNAs in antiviral immunity will change our understanding of RNA regulation and help to design new antiviral strategies.

| sRNAs in nematode-pathogen interactions
Plant-parasitic nematodes (PPNs) seriously threaten the safety of crop and agriculture production. PPNs can infect a variety of economically important crops like rice, wheat, maize, soybean, potato, tomato, and sugar beet. Over 4300 plant species from 197 genera have so far been reported as hosts of PPNs, and PPNs lead to over $150 billion losses in annual crops globally (Ali et al., 2017(Ali et al., , 2019. Recently, RNAi has been demonstrated in PPNs and shown to be influenced by sRNAs. It is known that miRNAs take part in plant-PPN interactions, for example different miRNAs are down-regulated to resist Heterodera schachtii, such as miR161, miR167a, miR164, miR172c, miR396a, miR396ab, miR396c, and miR398a (Hewezi et al., 2017;Khraiwesh et al., 2012).

| sRNAs function in biological control agent-induced systemic resistance
Plants have a complex network of interactions with many microorganisms. In addition to pathogens, there is also a class of beneficial  (Jiang, Fan, et al., 2020;Niu et al., 2016). Other studies found that Bacillus amyloliquefaciens FZB42 represses plant miR846 to induce systemic resistance via a JA-dependent signalling pathway (Xie et al., 2018).

| CROSS -K ING DOM RNA i IN PL ANT-MICROBE INTER AC TIONS
Previous studies showed that most sRNAs function endogenously during the interaction between plants and microorganisms. Recent evidence has shown that some sRNAs can move between the host cell and interacting organisms, and induce gene silencing via a mechanism called "cross-kingdom RNAi" (Huang et al., 2019).

Cross-kingdom RNAi was first demonstrated in plant-fungus inter-
actions (Weiberg et al., 2013). It found that Botrytis cinerea sRNAs (Bc-sRNAs) could hijack the host RNAi mechanism in Arabidopsis and tomato by binding AGO1 and silencing genes involved in immunity.
These fungal sRNAs represent a novel class of effectors that can inhibit host immunity with both DCL1 and DCL2 of B. cinerea. The dcl1/ dcl2 double mutant lost the ability to produce sRNA effectors and showed a significant reduction in pathogenicity (Zotti et al., 2018).
Since then, similar results have been reported and more sRNA effectors have been identified from other pathogens Wang, Weiberg, et al., 2017;Wang, Sun, et al., 2017). For example, B. cinerea delivers Bc-siR37 into the host cell to suppress immunity by targeting more than 15 genes, including receptor-like kinases, WRKY transcription factors, and cell wall-modifying enzymes. As a result, At-WRKY7, At-PMR6, and At-FEI2 exhibited enhanced disease susceptibility to B. cinerea (Wang, Weiberg, et al., 2017). Moreover, it was found that the Arabidopsis ago1-27 mutant was more resistant to Verticillium dahliae, which causes Verticillium wilt disease on many crops. Similar results have also been reported for B. cinerea.
These results indicate that V. dahliae also uses sRNAs to silence host target genes and which are associated with Arabidopsis AGO1 during infection . Puccinia striiformis f. sp. tritici also delivers a novel microRNA-like RNA1 (milR1) into wheat host cells and suppresses wheat pathogenesis-related 2 (PR2) gene in the defence pathway. Silencing of the milR1 precursor led to enhanced wheat resistance to P. striiformis f. sp. tritici (Wang, Sun, et al., 2017). In addition, cross-kingdom sRNA transport from microbes to hosts is not restricted to eukaryotic pathogens that encode RNAi machinery. For instance, the protozoan parasite Trypanosoma cruzi produces tRNAderived sRNAs that contribute to the ability to infect mammalian cells, although T. cruzi lacks canonical sRNA pathways (Garcia-Silva et al., 2014). Another study showed that the parasitic plant Cuscuta campestris can send miRNAs into host plants to silence host genes involved in the defence responses against C. campestris (Shahid et al., 2018). Additionally, the symbiotic bacterium Rhizobium delivers tRNA-derived sRNA fragments into soybean cells in an AGO1dependent manner, thus inducing plant nodulation-related gene silencing as in B. cinerea and V. dahliae (Ren et al., 2019).  . It was found that two miRNAs, miR159 and miR166, target the fungal gene isotrichodermin C-15 hydroxylase (VdHiC-15) and Ca 2+dependent cysteine protease calpain (VdClp-1), respectively . It was also shown that Arabidopsis plants can deliver siR-NAs, secondary phasiRNAs, into Phytophthora capsici, an oomycete pathogen, to induce the silencing of genes involved in pathogenicity .

| APPLI C ATI ON OF CROSS -K ING DOM RNA i IN CROP PROTEC TI ON
The ultimate goal of agricultural basic research is to transform new discoveries and advanced technologies into practical applications.
Host-induced gene silencing (HIGS) technology is an innovative concept of cross-kingdom RNAi technology that has emerged as a powerful alternative to chemical treatments for crop protection ( Figure 2). Numerous studies have demonstrated successful applications of HIGS technology in plants against a wide variety of plant diseases caused by pathogens such as viruses, viroids, bacteria, oomycetes, fungi, nematodes, and pests such as herbivorous insects, which cause significant economic loss (Coleman et al., 2015;Eschen-Lippold et al., 2012;Escobar et al., 2001;Fairbairn et al., 2007;Ghag, 2017;Govindarajulu et al., 2015;Huang et al., 2006Huang et al., , 2019Koch et al., 2013;Mao et al., 2007;Niu et al., 2021;Nowara et al., 2010;Panwar et al., 2018;Pooggin et al., 2003 (Nowara et al., 2010). Recent research has shown that HIGS is also effective in controlling necrotic fungal pathogens such as V. dahliae, B. cinerea, Puccinia triticina, and Fusarium species (Koch et al., 2013;Panwar et al., 2018;Wang et al., 2016). Down-regulation of syntaxin gene expression in potato by HIGS significantly suppressed Phytophthora infestans (Panwar et al., 2018). HIGS can also provide effective control of another oomycete disease, downy mildew disease, caused by Bremia lactucae, in lettuce. Transgenic lettuce lines expressing inverted repeats of fragments of either the HAM34 or CES1 genes of B. lactucae resulted in greatly reduced growth and inhibition of sporulation of the pathogen due to the specific suppression of these genes (Govindarajulu et al., 2015). In nematode disease control, HIGS technologies have also been reported recently (Fairbairn et al., 2007;Huang et al., 2006;Shivakumara et al., 2017). Silencing of two pharyngeal gland genes, msp18 and msp20, conferred transcriptional alteration of cell wall-modifying enzymes in Meloidogyne incognita and reduced nematode infectivity in eggplant (Shivakumara et al., 2017). In addition, HIGS of insect growth and development is a promising strategy for pest control in practice (Baum et al., 2007;Coleman et al., 2015;Mao et al., 2007;Zhang et al., 2015). Plant-mediated RNAi of MpPIntO2, MpC002, and Rack1 genes significantly decreased aphid population growth and reduced aphid reproduction by 40%-60% (Coleman et al., 2015). Taken together, all these examples illustrate that HIGS is a promising strategy to limit chemical-based pesticide applications.
Although HIGS is a promising technology, it relies on the generation of transgenic plants. Transgenic technology has not been successful in some crops, which limits the application of HIGS. Due to the lengthy and costly process of generating transgenic crops, an ecofriendly, non-genetically modified, RNAi-based crop protection strategy, spray-induced gene silencing (SIGS), has been developed.
SIGS is a potential, nontransformative, and environment-friendly pest and pathogen management strategy in which naked or nanomaterialbound dsRNA is sprayed onto leaves to cause selective knockdown of pathogenicity genes. It was found that spraying of dsRNA targeting fungal MoDES1 induced silencing of MoDES1 in M. oryzae and conferred efficient resistance against blast disease (Sarkar & Roy-Barman, F I G U R E 2 The application of transkingdom RNA silencing to plant disease resistance to pests and pathogens. 2021). In addition, SIGS approaches using the application of exogenous dsRNA can also suppress infection of Brassica napus by the pathogens B. cinerea and Sclerotinia sclerotiorum (McLoughlin et al., 2018). Similarly, RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs has been reported (Koch et al., 2016). Recently, it was reported that the efficacy of SIGS approaches is dependent on the RNA uptake efficiency of the pathogen (Qiao et al., 2021). To improve both RNA uptake efficiency and stability, current research efforts are focused on nanoparticle technology to improve the application system and the limited durability of the RNAi effect (Qiao et al., 2021). To facilitate the effective application of HIGS and SIGS, further studies will be needed to address the underlying mechanisms for cross-kingdom RNAi between plants and microbes.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
Collectively, many studies have highlighted the involvement of the Natural Science Foundation of Jiangsu Province (BK2021524).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created.