Role of Non-Coding RNAs in Plant Nutrition through Mycorrhizal Interactions

Abstract

In nature, many plants rely on symbiotic interaction with mycorrhizae for their nutrition and survival. For instance, nitrogen-fixing nodules and mycorrhizae are well established mutualistic biotic interactions between plants and bacterial/fungal partners under nitrogen limiting environment. Many small regulatory components of RNA like micro-RNAs play a critical role in establishment of these symbioses. These regulatory components are also crucial for balancing hormone levels, and synchronization of plant defenses and development pathways. However, functions of various sRNAs are still need to be addressed. This chapter will detailed out various important parts these regulatory components (sRNA, miRNA and siRNA) are playing during mycorrhizal interactions for plant growth, development and nutrition.

1. Introduction

During course of co-evolution since millions of years, plants have established symbiotic associations with the fungi and bacteria. Established mycorrhizal and rhizobia symbiosis with the plants are the best illustrated examples of such interactions. These symbiotic associations are entrenched by the molecular cross-talk including correct recognition and specific activation/repression of signaling pathways. Legume-rhizobia interactions are specific in terms of molecular cross talk, as the host plant secretes flavonoids which are perceived by compatible rhizobia for the induction, expression and activation of Nod genes in the bacteria, necessary for the nodule formation in the host plant. The secreted Nod factors once recognized by host specific intra-cellular kinase and extra-cellular LysM domain containing receptors, a cascade of cytoplasmic events starts within root epidermal cells. Depolarization of the membrane, alteration in calcium levels and induction of calmodulin based kinase signaling makes favorable environment for rhizobia infection thread formation and successive penetration of plant host cell through branching. Subsequently, ‘Bacteroids’ formation and nitrogen fixation initiates in host cell cytoplasm. In contrast, mycorrhizal interactions are not specific in terms of host range as they can colonize almost all terrestrial plants [1]. Although the signaling pathway for mycorrhizal symbiosis activation shares some attributes of rhizobial symbiosis events, induction and reprogramming of the host cells starts after the recognition of myc-LCO (mycorrhizal lipo-chito-oligosaccharide), which leads to altered metabolic cascade in host and hyphae as well [2]. This molecular cross-talk establishes nutrients and mineral transport through specialized and branched structures called ‘Arbuscules’ from Arbuscular mycorrhizal (AM) partner and photo-synthetically fixed carbon sources mobilization from host plant in exchange. To bear an invasion of microorganisms, plants must have some specialized mechanisms to distinguish beneficial microbes from harmful ones. Since last few decades, we are learning about regulators of fine tuning among symbiotic associations and plant immunity [3, 4]. Contributions of non-coding RNAs (ncRNAs) in this regulation of host defenses to establish symbiosis are indispensible according to recent studies [5].

In this chapter, we have summarized the genesis of various important classes of non-coding RNAs and their role in nutrient uptake, transport, assimilation and homeostasis in plants via mycorrhizal symbiosis, and discuss the recent discoveries of cross-kingdom RNA interference (RNAi) during plant-fungus interactions. We also provide the insights and future perspectives for improved understanding of mycorrhizal associations, which would aid in the development of innovative strategies for enhancing the crop yield.

2. Genesis of non-coding RNAs and classification

Currently, a large number of endogenously formed ncRNAs involved in different regulatory functions have been discovered and functionally characterized in various plant species [6, 7]. On account of their average size, the regulatory ncRNAs can be classified into sRNAs (small RNAs of typically 18–30 nt in size), medium-sized ncRNAs (broad range of 31–200 nt), and more than 200 nt sized Long-non-coding RNAs (lncRNAs). Furthermore, depending on their morphology, theycan be classified as linear or circular (circRNA). Recently, small regulatory RNAs (sRNAs), miRNAs and small interfering (si)RNAs, have been well characterized with respect to plant immunity and symbiosis. Although thought to be small, they play vital functions in response to the biotic, abiotic stress and environmental fluctuations by regulation/modulation of target genes expression [8, 9, 10, 11]. Similarly, lncRNAs were considered transcriptional noises, but later attracted attention for the heterogeneous groups of ncRNAs and long range [12]. Remarkably, unlike other linearly regulated ncRNAs, the newly discovered circRNAs belong to a novel class, which lacks free 5′ and 3′ end [13]. In addition, many small ncRNAs, which are derivatives of tRNAs, which are identified and characterized in plants typically comprised of 15–42 nt, termed as tsRNAs [14, 15, 16]. The tsRNAs are also classified as regulatory ncRNAs for multiple functions. Generally, the functions of certain ncRNA are similar, but some differ and overlap in silencing signaling pathways [17].

As reviewed by Chao et al. [18], the biogenesis of miRNAs is a multi-step procedure which involves transcription, processing, alterations, and then RNA-induced silencing (RISC) complex assembly. First, a pri-miRNA (primary miRNA) is transcribed from RNA Polymerase II containing a hairpin RNA secondary structure. Next, the base pri-miRNA hairpin is then cleaved by a DICER Like RNase-III family enzyme (usually DCL1). To release miRNA-miRNA* duplex, these hairpins are cleaved again and subsequently methylated (at 2′O- position) by HUA Enhancer 1 (HEN1) nuclear protein for the stability. Finally, in nucleus the mature miRNA strand enters into AGO1 to form miRNA-AGO1 complex, which are then transported to cytoplasm leaving behind cleaved miRNA* fragment for the induction of post-transcriptional gene silencing.

Depending on their mechanism of action, siRNAs can further be classified into three major sub-categories: (1) native antisense siRNAs (nat-siRNAs), (2) heterochromatin siRNAs (hc-siRNAs), and (3) trans-acting siRNAs (ta-siRNAs). ta-siRNA is generated from the TAS gene which is transcribed from RNA Pol II into single-stranded RNA and loses its cap and poly-A tail during miRNA-AGO1 complex-controlled cleavage [8, 19]. Later, the 5′ or 3′ cleaved fragments are end protected by the suppressor of gene silencing 3 (SGS3) protein and transformed into double-stranded RNA (dsRNA) via RDRP-VI [20]. Finally, by HEN1 and DCL activities they are methylated and processed to form ta-siRNAs (21–24 nt). To participate in post-transcriptional modulation/silencing of target genes by pairing with its complementary mRNAs, these 21–24 nt sized strands are integrated with AGO1/AGO7 present in the cytoplasm, whereas a few ta-siRNAs are loaded onto AGO4/6 for assisting methylation of TAS genes via RNA Pol V.

tsRNAs, with a wide size range (15–42 nt), represent a unique ncRNA class that can be sub-categorized based on their cleavage sites: (1) tRF-1 s, (2) tRF-2 s, (3) tRF-3 s, (4) tRF-5 s, and (5) tiRNAs. However, plant research is still in its infancy and many questions remain unanswered in reference to its existence. For instance, the biosynthetic pathway for tsRNAs and their regulatory or physiological roles in plants are still very limited [21].

CircRNAs are known as RNA biomolecules which are circular, covalently closed and single-stranded [22]. They were first identified and characterized from plant viruses by Sanger and colleagues in 1976. The organization of circRNAs can be divided into three groups [23]. (1) The Exon circRNAs are generated by the circularization of lariat-derived and intron pairing, (2) the intronic circRNAs are formed by the partial intron degradation after lasso structure formation; and (3) exo-syntronic circRNAs are composed of exons as well as introns, and circularized during the splicing process.

lncRNAs biogenesis can be categorized into five major types in accordance to the sites being transcribed via RNA-Pol II. (1) The antisense lncRNA is transcribed over the complementary strand of the exon; whereas (2) sense lncRNA is transcribed on the same strand as the exon. As name indicates, (3) Intron lncRNA is transcribed into an intron. (4) The Inter-genic lncRNAs are situated between two different genes and (5) the enhancer lncRNA mostly arises from the enhancer region of the protein-encoding gene [24]. They can control target regulation in a variety of ways, including chromatin re-modeling, transcriptional repression, splicing of RNA and its transcriptional enhancers. Additionally, lncRNAs can code for certain small peptides required for various cellular processes [25]. Notably, several lncRNAs are regulated under abiotic/biotic stresses in the plants.

3. Role of sRNAs in plant nutrition

Induction of miRNAs regulates the expression of an array of genes and promotes plant nutritional homeostasis. Owing high-throughput RNASeq techniques and target prediction tools the role of ncRNAs in nutrition and stress signaling has been investigated in recent past. Majorly the role of ncRNAs in nitrogen (N), phosphate (Pi) and sulfur (S) homeostasis has been discussed below:

3.1 Nitrogen

Evidence for miRNAs controlling nitrogen responses in plants has been illustrated [26, 27]. Up-regulation of pri-miRNA156, pri-miRNA447c and down-regulation of pri-miRNA169 and pri-miR398a has been characterized in Arabidopsis under nitrogen-deficient conditions [28]. Expression of nitrogen responsive miRNA, like miRNA160, miRNA167, miRNA168 in the maize roots and miRNA164, miRNA171 in shoots whereas, miRNA169 in both are reported under nitrogen-limiting conditions [27]. Similarly, several nitrogen-responsive miRNAs have been investigated in legumes, for instance, a total of altered expressions of 168 miRNAs are reported in limiting-nitrogen-tolerant and limiting-nitrogen-sensitive genotype of soybean using RNASeq [26]. A down-regulation of miRNA2606a/b-3p and up-regulation of miRNA1512a-5p was found in limiting-nitrogen-tolerant and limiting-nitrogen-sensitive genotype respectively. Moreover, mRNA transcripts encoding Cathepsin and E3-Ubiquitin ligase protein were found to be targeted and degraded by miRNA396b/g-5p and miRNA156b/6f-5p respectively under nitrogen stress.

3.2 Phosphate

Phosphate-responsive sRNA involved in Pi-uptake, transport, assimilation and homeostasis through targeting mRNA transcripts are extensively studied and identified in plant including rice, maize, tomato, soybean and Arabidopsis [29, 30, 31, 32, 33]. Among these plant species, common set of plant miRNA families are characterized modulating signaling networks, including miRNA156, miRNA159, miRNA166, miRNA319, miRNA395, miRNA398, miRNA399, miRNA447, and miRNA827 are demonstrated in response to Pi-limiting environment [34, 35]. An elevated level of miRNA156, miRNA399, miRNA778, miRNA827, miRNA2111 and suppressed miRNA169, miRNA395 and miRNA398 levels are observed under Pi stress [28]. Role of miRNA2111 has been illustrated under N and Pi limiting conditions [36]. The expression of phosphate-responsive PHO2 transporter was altered by miRNA827 and miRNA399 [37]. Moreover, miRNA827 targets the Major Facility Superfamily (MFS)-XPS proteins which are involved in Pi sensing and transport [38]. Common response against nutrient starvation includes anthocyanin accumulation in plants. MYB TF regulated anthocyanin biosynthesis pathway genes are targeted by siRNAs produced by ta-siRNA4 under the regulation of Pi-responsive miRNA828, post-transcriptionally [39]. The major regulatory role in maintenance of mineral homeostasis in host plant under N, Pi and C limiting environments is performed by miRNA398a [40]. Among all the characterized Pi-responsive miRNAs, altered levels of different alleles of miRNA399 were found conserved and pre-dominant under Pi-limiting conditions [41]. miRNAs and siRNAs induction was reported upon Candidatus liberibacter infection in citrus plants and interestingly, miRNA399 level was found elevated in infected plants than healthy host under Pi-limiting conditions [33]. These facts demonstrate the critical role of sRNAs in post-transcription regulation of Pi-responsive transcripts enabling host adaption under nutrition stress.

3.3 Sulfate

Sulfate transporters located on root epidermal and cortical cell membrane are the key components in sulfur uptake and transport to the plants in SO4²⁻ form. Based on their substrate affinity, sequence similarity and their location of expression, they are categorized into five major groups. Group 1, group 2 and group 3 are characterized as high affinity, low affinity, and moderate affinity transporters for sulfur substrate, respectively [42, 43, 44]. Group 4 and 5 transporters are characterized as efflux transporter on tonoplast and molybdenum transporters (for being actively involved in molybdenum transport across the plant) respectively [45, 46]. S uptake from soil to the root is carried out by group1 and 2 transporters, while root to shoot transport of S is done by group 4 transporters. Under S-limiting conditions plethora of miRNAs induced including miRNA66, miRNA67 and miRNA395 while suppression of miRNA14, miRNA20 and miRNA43 is associated with regulation of post-transcription modification of S signaling. miRNA395 is a S-specific ncRNA signal and has been characterized to function as a key regulator of the sulfate depletion pathway. Under S-deprivation, miRNA395 positively regulates the expression of the low-affinity transporter AtSul2;1 [47], supporting sulfate uptake and transport of cells to shoots and leaves in Arabidopsis thaliana. The initial step of S assimilation into cysteine is catalyzed by ATP dependent sulphurylases (APSs), which are the target for miRNA395 in plastids [35].

4. Nutrient uptake/exchange during mutualistic plant-fungus interactions

One of the characteristics of the beneficial mycorrhizal interactions is the bidirectional nutrient exchange between both the partners and to support the growth of plant host [48, 49]. In these relationships, the fungus provides nitrogen, phosphate and sulfate nutrients to the host, whereas, in return, the host plant transfers photosynthetically fixed carbon (4–20%) to the mycorrhizal fungus [50]. In AM roots, the fungus proliferates into the root cortex intercellularly as well as intracellularly, whereas in case of ECM roots, it only covers intercellular regions, indicating the differences in the mechanism of colonization and nutrient uptake/exchange. The uptake of nutrients by plants from soil is limited by the repressed mobility of nutrients. Importantly, during AM symbiosis, the plant phosphate (P) transporters are down-regulated [51, 52]. Under these conditions, the nutrient such as P uptake is predominantly achieved through mycorrhizal pathway [1, 53, 54]. It has been observed that contribution of the mycorrhizal pathway in nutrient uptake varies with the plant and fungal partners that are involved in the interaction and also on the nutrient type [48, 52, 55, 56, 57]. To facilitate the nutrient uptake via mycorrhizal interface, the peri-arbuscular membrane (PAM) harbors high affinity transporters that are specifically induced in mycorrhizal roots. For instance, Pt4 and AMT2 are the high affinity transporters for P and ammonium (NH4⁺) that mediate transfer of respective nutrients from fungus to plant host [58, 59, 60]. Moreover, a few mycorrhiza-inducible sulfate transporters have also been reported in AM roots [61, 62]. Recently, a sulfate transporter (SiSulT) and iron transporter (PiFtr) from Serendipita indica (previously known as Piriformospora indica) has been characterized, which transfers sulfur to the maize and iron to the rice plants, respectively and improves its growth [63, 64]. These studies highlight the importance of sulfur transport via mycorrhizal associations.

On the other hand, the plant transfers photosynthates as sucrose from source to ECM roots that serve as a carbon sink, which is then converted to simpler sugars such as glucose or fructose by invertase enzyme of host. The glucose and fructose are taken up by fungal counterparts through mycorrhizal interface. For instance, an arbuscular membrane localized monosaccharide transporter (MST2) is involved in the uptake of glucose and xylose molecules by AM fungi [65]. Intriguingly, host carbon supply has been found to trigger the fungal gene expression, and P and N uptake during AM and ECM symbiosis [66, 67, 68]. This also leads to increased hydrolysis of polyP (an important source of P and N) and release of Pi and Arg in the fungal cytoplasm. The Arg is further broken down to NH4⁺ and is transferred to host plant via mycorrhizal interface. Importantly, these transport processes, from host to fungus and from fungus to the host, involve diverse molecular players that mediate membrane transport for the nutrition exchange between mycorrhizal plants and fungus at the interaction interface. The membrane transporters and channels that mediate the transport of molecules such as P, N, S, K, sugar and water are collectively referred as the ‘transportome’ [69].

Moreover, several robust and tightly regulated signaling processes are involved in establishing the successful mycorrhizal colonization for the efficient exchange of nutrients between plant and fungus. Although, the regulatory processes of plant and fungus both are important during symbiosis, the major proportion of studies has focused on the regulation from plant’s perspective. These regulators include a variety of non-coding RNAs, phytohormones, peptide signals, transcription factors such as CYCLOPS and NODULATION SIGNALING PATHWAY (NSP1 and NSP2) [70, 71, 72]. The CYCLOPS, NSP1 and NSP2 are conserved members of rhizobial and mycorrhizal symbiosis phenomenon. The detailed overview of transcriptional regulation of AM development has been provided by Pimprikar and Gutjahr [73]. The non-coding RNA mediated-regulation of mycorrhizal symbiosis is now gaining the scientist’s attention and emerging as new area of research.

5. Plant’s ncRNAs modulating interconnection networks with fungi

5.1 miRNA

In recent past decades, the focus has pushed beyond the traditional defense pathway’s transcriptional control in establishing pathogenic or beneficial plant-microbe interactions to attempts to understand novel transcriptional regulators. In particular, many groups have started investigating the function of microRNAs (miRNAs) in regulating signaling processes accompanying the symbiotic interactions. Many plant miRNAs have been reported to be involved in modulating the plant-pathogenic microbe interactions. The majority of miRNAs investigated and characterized to date complements the alterations described well in the transcriptome. For example, one of the defenses against necrotrophic pathogens involves improving the physical tolerance of plant cells. Arabidopsis miRNA408 and miRNA160a induce physical cell reinforcement by positive regulation of lignification and callose deposition [74, 75]. In Medicago and Oriza, various miRNA targets the ET (Ethylene), JA (Jasmonic Acid), and SA (Salicylic Acid) biogenesis during pathogenesis at very early stages [76, 77]. During attack by biotrophic pathogens, many miRNAs also suppress routine cellular detoxification. One example is miRNA398, which enhances ROS generation in Magnaporthe oryzae-infected tissues [78]. On the other hand, in case of tomato, the necrotrophic association between plant and Alternaria solani, miRNAs target gene transcripts are reported to be actively involved in toxin detoxification [79]. This suggests that miRNAs have potential to modify plant cells into the toxic ecosystems, poisoning invading microbes before they spread further to strengthen host immunity.

In mutual bio-trophic interactions, a few recent investigations have shown that the a major proportion of miRNAs synthesized all through interaction establishment modulate hormone response pathways, protein methylation, and functions of innate immunity components [78, 80]. A well-studied example includes the miRNA (annotated as E4D3Z3Y01BW0TQ) which is reported to be induced during AM symbiosis progression and interferes with GA signaling. GA signaling pathway is known to inhibit symbiotic-association [81, 82, 83]. On the other hand, miRNA172c promotes nodule formation in many plants by repressing the translation of APETALA2 TF [84, 85]. In AM-colonized roots, miRNA171b hampers with GRAS TF-responsive transcripts targeting through miRNA171, which are necessary for both nodulation and symbiosis.

5.2 siRNA

Microorganisms can have a significant impact on how plants react to colonization; they are not just background actors in the process. Since past few decades, effector proteins and siRNAs have been the two main areas of research. A variety of plant signaling pathways are altered by microbial effector proteins, which are generally tiny secreted proteins that are substantially stimulated during the colonization process. Similar to effector proteins, siRNAs target essential plant transcripts, disrupt transcripts via the ARGONAUTE (AGO) pathway, or act in a manner resembling that of miRNAs by inhibiting tRNA binding and localization. The microorganism-secreted siRNAs are taken up by the host plant, and disrupt the key transcripts of host.

Botrytis cinerea, a fungal pathogen, has been demonstrated to alter plant physiology during colonization by secreting siRNAs [86]. B. cinerea siRNAs initially penetrate host plant cells during the pathogenesis of tomato and Arabidopsis, where they diminish the host’s RNAi apparatus. These relatively tiny molecules can therefore be thought of as variants of conventional effector proteins. In case of A. thaliana, Bc-siRNA3.1, Bc-siRNA3.2, and Bc-siRNA5 collectively silenced the stress-related genes, such as PRXIIF, WAK, MPK1 and MPK2 to eliminate the plant defense [87]. However, Bc-siRNA37 AtPMR6, AtFEI2 and AtWRKY7 are selectively silenced by Bc-siRNA37 [88]. While this is the only example so far in which siRNAs have been formed and released by microbes and reached to the host plant cells, there can be another common means based on genomic analysis by which microbes can modulate host responses during colonization.

5.3 lncRNA

The role of lncRNAs has been well-investigated in the context of plant defense against fungal, bacterial and viral pathogens [89, 90, 91, 92, 93, 94]. Furthermore, the functions of a number of plant lncRNAs intricated in plant defenses are experimentally validated. For instance, it has been observed that the lncRNA-ACOD1 is dispensable for viral entrance but not for viral replication in the host [95]. Moreover, when turnip crinkle virus infection occurs in Arabidopsis, the expression of the long intergenic ncRNA LINC-AP2 gene is negatively regulated [89]. LncRNA33732 has been characterized to function as a positive regulator in tomatoes, enhancing the expression of the respiratory burst oxidase gene and raising H₂O₂ build-up, thereby increasing tomato resistance to Phytophthora infestans [96]. Also, lncRNA23468 in tomato can compete with endogenous RNA to regulate the NBS-LRR gene by feeding on miRNA482b, thereby controling tomato resistance to P. infestans pathogenesis [90]. Numerous lncRNAs have been identified as being modulated in drought, nitrogen-stress and phosphate depletion in maize [97, 98, 99]. The maize inbred line B73 tissues were subjected more than 700 high-fidelity RNA-Seq studies, which identified nearly 18,165 maize lncRNAs [100]. Although lncRNAs are involved in the regulation of plant-microbe interactions, there are no available reports characterizing lncRNA responses to AM fungi, so far as the experimental evidence is considered.

6. miRNA in symbiosis: regulation through repression

Emphasis on existence and importance of small ncRNA, especially miRNA started with the discovery of its association with regulation of gene expression in Petunia [101, 102]. Owing genome wide studies and high-throughput sequencing efforts, till date thousands of miRNAs have been characterized throughout the kingdom of life. Most of the characterized miRNA associated with symbiosis are either involved in nutrient signaling, exchange and homeostasis or development of nodule/arbuscules or both [103, 104] are illustrated in Figure 1. Based on morphological analysis of host, AM Symbiosis (AMS) can be sub-categorized into four major stages: (1) pre-contact signaling, (2) contact establishment between plant root and fungal hyphae, 3) intra-radical proliferation, and 4) arbuscule formation [105]. A non-canonical form of miRNA171, which is found repressed under phosphate starvation, regulates an important transcription factor (TF) involved in common symbiotic signaling pathway, NSP1/2 (NODULE SIGNALING PATHWAY 1/2) during mycorrhizal symbiosis.

Figure 1.

Illustration of cross-talk between myc-LCO (mycorrhizal lipo-chito-oligosaccharide) factor induced common pathway signaling, miRNAs and regulatory hormones during AM symbiosis. Solid and dotted lines represents direct and indirect interconnections respectively.

The interaction of NSP2 with the mycorrhizal-specific GRAS TF, RAM1 (Required for Arbuscular Mycorrhization 1) regulates RAM2, and DWARF27 that are part of cutin and strigo-lactone biosynthetic pathway, respectively, as these two pathways facilitate AM inoculation. miRNA393, also identified as a N- and Pi-responsive miRNA, is involved in the homeostasis of auxin signaling and thus inhibits the auxin receptor TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/ABF), also mediates the repression of root growth regulatory factors (GRFs) to affect fungal colonization and arbuscule development. miRNA396b also investigated to perform a significant role in root colonization and development during mycorrhizal symbiosis by targeting six GRFs and a TF in Medicago truncaluta [106].

For initiating rhizobia symbiosis, interactions between miRNAs, Nod factor signaling, and hormone regulation through NSP2 is controlled by a nodule-specific miRNA171. The NUCLEAR FACTOR (NF)-YA gene, a TF necessary for the nodule initiation and maintenance of meristem, is negatively regulated by miRNA169 [107]. The combination of cytokinin-responsive miRNA172 genes, Nod factor, and various AP2 (APETALA2) targets has been implicated in rhizobia infection, stimulation of nodule organogenesis, N2 fixation, and delaying senescence in nodule cells.

The auto-regulation of the nodulation (AON) pathway, which governs the number of nodules that are formed in host plants, is one mechanism by which miRNA172 can work. In this pathway, leucine-rich repeat receptor-like kinase (LRR-LRK) receptors recognize NF- or nitrate-induced Clavata3/Embryo Surrounding Region-Related-peptides, resulting in an inhibitory signal (including CK) to cells, for establishing new nodules. By suppressing certain squamosa promoter-binding protein-like (SPL) TFs that stimulate miRNA172 production, miRNA156 antagonizes the action of miRNA172 [108].

Through the repression of nodulin gene (specifically ENOD93), miRNA393j-3p restricts nodules [109] whereas, miRNA1512 and miRNA1515 over-expression was discovered to be linked to increased nodule formation [75]. Finally, miRNA160 and miRNA167 cleave the transcripts of multiple auxin response factors (ARFs) that play key roles in the auxin response and pre-requisite for nodule initiation [110]. The detailed regulation mechanism is shown in Figure 2. miRNA390encourages the formation of a transacting small interfering (tasi)RNA, that represses ARF3 and ARF4during rhizobia colonization and nodule growth [78], it is known to combine auxin and ethylene signals. While, ARF10, ARF16, ARF17 and ARF6, ARF8 are directly regulated/targeted by miRNA160 and miRNA167, respectively [111] for auxin-responsive root development in both cases.

Figure 2.

Illustration of cross-talk between Nod factor induced common pathway signaling, miRNAs and regulatory hormones during rhizobial symbiosis. Solid and dotted lines represents direct and indirect interconnections respectively.

7. Mycorrhiza-derived non-coding RNAs and cross-kingdom signaling during symbiosis

Recent investigations have established non-coding RNAs as one of the central mediators of cross-kingdom communication between the plant and microbes [86, 88, 112, 113, 114, 115, 116, 117, 118, 119]. These non-coding RNAs can move from donor organism to recipient organism, and target the specific host mRNAs for degradation. Sometimes sRNAs also trigger the production of secondary sRNA and thereby modulate the host defense and metabolic pathways [87, 120, 121]. Most of the studies focus on the plant-parasite or plant-pathogen (fungi and oomycetes) interactions [86, 88, 112, 120], however, such processes have been rarely explored in case of plant-mycorrhiza associations. Emerging body of evidences suggest that many plant miRNAs show differential expression patterns during AM symbiosis, nevertheless, their functions and cross-kingdom mobility remains unclear [80, 83, 111, 122, 123]. Mewalal et al. [124] identified several sRNAs from Polpulus spp. which were responsive to mutualistic/symbiotic interaction with mycorrhizal fungi like Laccaria bicolor and Rhyzophagus irregularis. Interestingly, they did not find any Populus RNAs interacting with R. irregularis, however, some of the miRNAs could interact with L. bicolor. Further the study revealed that these miRNAs can potentially target multiple host mRNAs encoding for vesicular transport and transcription regulatory proteins along with several uncharacterized proteins.

On the other hand, at present, very little information is available regarding the non-coding RNA biogenesis machinery and their functions in the development of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi or while interacting with the host plants. However, the successful application of host-induced gene silencing (HIGS) and virus-induced gene silencing (VIGS) approaches [65, 125, 126, 127, 128] indicates that AMF, like pathogenic fungi, also possess functional RNAi machinery. An in silico study identified putative RNAi machinery including a Dicer-like (DCL) gene, Argonaute-like (AGO-like) and RNA-dependent RNA polymerase (RdRp) gene families in R. irregularis, and validated their transcript-level expression [129]. An unsual expansion of AGO-like (5 members) and RdRp (21 members) gene families was observed in R. irregularis. Authors postulated that 15 out of 21RdRp genes, could be the product of a recent gene expansion event. The study also characterized the fungal sRNA and microRNA-like sequences, and predicted 237 transcripts of Medicago truncatula as their potential targets including a few known mRNAs that are modulated during AMF colonization. For instance, some of the M. truncatula mRNAs that are potentially targeted by Rir-sRNAs encode for the nuclear-binding leucine-rich repeat (NBS-LRR) type disease resistance gene, Non-specific phospholipase C4 (NPC4), MtVapyrin (Ankyrin repeat RF-like protein) and DREPP plasma membrane protein (MtDREPP) [129]. The homologs of NBS-LRR and NCP4 proteins from rice and arabidopsis, respectively, are involved in the plant immunity [130, 131], thus repression of these genes may allow AM colonization without triggering the robust host defense responses. MtVapyrin plays crucial role in arbuscule formation [132, 133, 134]. The down-regulation of MtDREPP has been reported in mycorrizal roots [135]. Though, further experimental validation is required, these findings indicate the possible existence of non-coding RNA-mediated post-transcriptional regulation and cross-kingdom gene silencing by AMF.

Another study by Silvestri et al. [136] identified the small RNA population from AMF Gigaspora margarita and showed their origin from different genetic sources such as endobacteria, RNA viruses and non-integrated DNA sequences from mitoviruses. Intriguingly, the extracellular vesicles (EVs), that are deployed in delivering the sRNA molecules to the other interacting partner [112, 120, 137], have also been observed in the peri-arbuscular interface of R. irregularis during the whole lifespan of arbuscules. This indicates the crucial role of EVs in cross-kingdom communication and nutrient exchange during AMF symbiosis [138]. More recently, a breakthrough discovery demonstrated that an ECM fungus Pisolithus microcarpus encodes 11 miRNAs, six of them were found induced during host colonization process. Notably, the miRNA (Pmic_miR-8) enters the plant cell and partakes in cross-kingdom gene silencing at some stage in symbiotic interaction with host plant Eucalyptus grandis [139]. The inhibition of Pmic_miR-8 resulted in less developed Hartig nets, whereas, supplementation showed increased Hartig net depth in host tissue. Further the study showed that Pmic_miR-8 may target the host NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain containing transcripts, indicating its potential role in modulating host signaling to stabilize the mutualistic association. As the CC (coiled-coil) nucleotide binding and leucine-rich repeat domain immune receptors (CC-NLR) are the largest category of NLRs, thus Pmic_miR-8 may target several plant genes belonging to this class. Importantly, this is the first study which established the cross-kingdom gene silencing by mycorrhizal fungi and its role in beneficial interactions with host.

8. Conclusion and future prospects

In the last decade, the non-coding RNAs have emerged as one of the key regulators of diverse plant process including their development, response to abiotic/biotic stress, and nutrient uptake. A significant advancement has been made to understand the crucial roles of non-coding RNAs in plant-microbe interactions, particularly pathogenic interactions. Nutrient uptake via mycorrizal association is an important aspect of plants lifestyle and the studies suggest the extensive involvement of non-coding RNAs in regulating the plant nutrient status via affecting symbiosis. Notably, the sRNA-mediated regulatory mechanisms during mycorrhizal symbiosis have mainly focused on plant’s perspective. These studies have provided crucial insights on understanding how the mycorrizal colonization proceeds and how the host plants fine-tune the extent of fungal colonization so that it does not turns pathogenic. On the contrary, the sRNA-mediated regulation of symbiosis from the fungal perspective remains infancy. Moreover, an integrated view of both the organisms (plant and fungus) will be required to appropriately comprehend the beneficial relationships. To understand ncRNA-molecular interaction networks occurring at plant host-AM symbiosis interface, experimental evidences and rewriting of dynamics of interaction, sensing, uptake, transport, assimilation and homeostasis of nutrients regulation are required. Extensive investigations for ncRNAs mediated regulation of cross-talk between AM and host plant are also needed for teeming knowledge voids. We anticipate that the recent discovery of cross-kingdom gene silencing by ECM fungus Pisolithus microcarpus and AM-like model EM fungus Serendipita indica would pave the way for future investigations of non-coding RNA-mediated regulatory networks in mycorriza growth and development as well as during host interactions, and would trigger novel research ideas among plant scientists. The better understanding of these regulatory circuits would aid in improving the nutritional status of plants in order to combat the elevating global quality-food demand.