**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.

**Keywords:** miRNA, siRNA, mycorrhiza, nodulation, symbiosis, nutrient uptake

### **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 [8–11]. 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 [14–16]. 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 posttranscriptional 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 complexcontrolled 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 doublestranded 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 posttranscriptional 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 proteinencoding 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.
