**1. Introduction**

Small RNAs (sRNA) are small noncoding RNA segments of 19–30 nucleotides in length [1]. They mediate gene silencing, a gene regulation mechanism acting on a transcriptional

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(transcriptional gene silencing (TGS)) and post‐transcriptional level (post‐transcriptional gene silencing (PTGS)). In general, sRNA molecules originate from the transcription of endogenous microRNA (miRNA genes), other genomic sRNA loci, aberrant RNA produced by transposons as well as invasive viral RNA [2]. Plants carry two main classes of sRNAs grouped according to their size, function and biogenesis, namely microRNAs (miRNA) and short‐interfering RNAs (siRNA) [3]. Such sRNAs are generated through various mecha‐ nisms; within the miRNA biogenesis pathway, miRNA precursors derived from MIR genes are processed in the nucleus by Dicer-like protein 1 (DCL1) and exportin-like protein (HYL1) into mature miRNA duplexes of 20–22 nucleotides in length. Mature miRNAs are then meth‐ ylated at the 3′ terminus by HEN1 (small RNA methyltransferase) and exported to the cyto‐ plasm. One strand of the duplex is incorporated into an argonaute protein (AGO) protein to form an RNA‐induced‐silencing complex (RISC) [4]. The siRNAs, however, originate from long dsRNA that can be derived from transgenes, viruses, transposons and natural senseantisense transcripts. Such long dsRNA is recognized and cleaved by a certain type of DCL proteins; thereby siRNA classes with different sizes are generated. Like miRNAs, siRNAs are loaded into an AGO protein-containing RISC that controls gene expression patterns through the degradation of mRNA or the repression of translation of fully/partly complementary sequences of mRNAs, as well through epigenetic changes via mediation of DNA and histone methylation [5, 6].

Gene silencing is not only important for the maintenance of genome integrity by silenc‐ ing transposons or by degrading the viral RNA but also important during host immune responses of both plants and animals [7–9]. The recognition of pathogens by plants leads to the activation of a multi-layered immune system that comprises the establishment of a complex network of inducible defences including pathogen-associated molecular pat‐ tern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) [10, 11]. The entire signalling process involves the regulation of defence gene expression, the release of plant hormones and/or the induction of secondary metabolites [12]. Over the past few years, plant sRNA pathways were recognized as important players during PTI and ETI [13, 14]. In Arabidopsis, bacteria-induced miRNAs were identified to orchestrate com‐ ponents of plant hormone signalling, including auxin, abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) [15, 16]. A canonical example of an miRNA regulating plant defence is miR393. It is up-regulated upon treatment with a bacterial PAMP, and nega‐ tively regulates auxin signalling and therefore contributes to SA-mediated PTI responses in Arabidopsis [17].

Although the important role of sRNAs in plant defence against viruses and bacteria is documented [8, 13], their function as components of the plants' defence response against fungi is less clear. Advances in genome‐wide studies revealed a massive adaptation of host miRNA expression patterns after infection by fungal pathogens such as *Fusarium vir‐ guliforme* [18], *Erysiphe graminis* [19], *Verticillium dahliae* [20], *Cronartium quercuum* [21], as well as the oomycete *Phytophthora sojae* [22]. The alterations in sRNA expression profiles upon fungal attack suggest that gene silencing also contributes to antifungal defence; how‐ ever, up to date there are no putative mechanisms deciphered. Besides orchestrating plant defence, sRNA could also act as direct antifungal molecules, as some plant miRNAs could share complementarity to fungal genes. This possibility has already been demonstrated by engineering transgenic plants expressing dsRNA targeting fungal genes and exhibiting enhanced resistance to different fungi. For instance, this mechanism named host-induced gene silencing (HIGS) was successfully applied for various plant-fungi pathosystems such as silencing of the *Blumeria graminis* effector Avra10 [23], or *CYP51* genes of *F. graminearum* [24].

(transcriptional gene silencing (TGS)) and post‐transcriptional level (post‐transcriptional gene silencing (PTGS)). In general, sRNA molecules originate from the transcription of endogenous microRNA (miRNA genes), other genomic sRNA loci, aberrant RNA produced by transposons as well as invasive viral RNA [2]. Plants carry two main classes of sRNAs grouped according to their size, function and biogenesis, namely microRNAs (miRNA) and short‐interfering RNAs (siRNA) [3]. Such sRNAs are generated through various mecha‐ nisms; within the miRNA biogenesis pathway, miRNA precursors derived from MIR genes are processed in the nucleus by Dicer-like protein 1 (DCL1) and exportin-like protein (HYL1) into mature miRNA duplexes of 20–22 nucleotides in length. Mature miRNAs are then meth‐ ylated at the 3′ terminus by HEN1 (small RNA methyltransferase) and exported to the cyto‐ plasm. One strand of the duplex is incorporated into an argonaute protein (AGO) protein to form an RNA‐induced‐silencing complex (RISC) [4]. The siRNAs, however, originate from long dsRNA that can be derived from transgenes, viruses, transposons and natural senseantisense transcripts. Such long dsRNA is recognized and cleaved by a certain type of DCL proteins; thereby siRNA classes with different sizes are generated. Like miRNAs, siRNAs are loaded into an AGO protein-containing RISC that controls gene expression patterns through the degradation of mRNA or the repression of translation of fully/partly complementary sequences of mRNAs, as well through epigenetic changes via mediation of DNA and histone

Gene silencing is not only important for the maintenance of genome integrity by silenc‐ ing transposons or by degrading the viral RNA but also important during host immune responses of both plants and animals [7–9]. The recognition of pathogens by plants leads to the activation of a multi-layered immune system that comprises the establishment of a complex network of inducible defences including pathogen-associated molecular pat‐ tern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) [10, 11]. The entire signalling process involves the regulation of defence gene expression, the release of plant hormones and/or the induction of secondary metabolites [12]. Over the past few years, plant sRNA pathways were recognized as important players during PTI and ETI [13, 14]. In Arabidopsis, bacteria-induced miRNAs were identified to orchestrate com‐ ponents of plant hormone signalling, including auxin, abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) [15, 16]. A canonical example of an miRNA regulating plant defence is miR393. It is up-regulated upon treatment with a bacterial PAMP, and nega‐ tively regulates auxin signalling and therefore contributes to SA-mediated PTI responses

Although the important role of sRNAs in plant defence against viruses and bacteria is documented [8, 13], their function as components of the plants' defence response against fungi is less clear. Advances in genome‐wide studies revealed a massive adaptation of host miRNA expression patterns after infection by fungal pathogens such as *Fusarium vir‐ guliforme* [18], *Erysiphe graminis* [19], *Verticillium dahliae* [20], *Cronartium quercuum* [21], as well as the oomycete *Phytophthora sojae* [22]. The alterations in sRNA expression profiles upon fungal attack suggest that gene silencing also contributes to antifungal defence; how‐ ever, up to date there are no putative mechanisms deciphered. Besides orchestrating plant defence, sRNA could also act as direct antifungal molecules, as some plant miRNAs could

methylation [5, 6].

10 Plant Engineering

in Arabidopsis [17].

In this study, we aim to elucidate the role of sRNAs in regulating susceptibility to *Colletotrichum* spp.; hence we congregated results from two compatible pathosystems: *C. higginsianum*, which infects plants from the Brassicaceae family such as *Arabidopsis thaliana* (**Figure 1**) and *C. graminicola*, which is a devastating pathogen of the industrially important crop *Zea mays* (**Figure 2**). Both ascomycetes use a multistage hemibiotrophic strategy to infect their host and also share close genetic similarities making them tractable models to compare fungal pathogenicity in both dicot and monocot models [25–28]. In Arabidopsis, *C. higginsianum* employs first a biotrophic stage limited to a confined array of first invaded cells, from where the fungus develops secondary hyphae to switch to necrotrophic growth into surrounding cells. *C. graminicola* extends the biotrophic lifespan into many host cells, persisting biotrophic at the margins, whereas the centre of infection becomes necrotrophic. *C. graminicola* is a major worldwide threat for corn cultures, as it affects all parts of the plants, either as leaf blight or as stalk rot [29]. Depending on specific corn hybrids and culture conditions, *C. graminicola* can result in up to 40% yield loss where endemic.

During the first step, a selection of sRNA mutants and two fully and intermediate fungal sus‐ ceptible accessions of *A. thaliana* was examined in order to dissect possible defence defects caused by mutations in sRNA biogenesis pathways. Thus, we analysed the accumulation of phytohormones that are known to mediate Arabidopsis resistance against *C. higginsia‐ num* [30] and secondary metabolites that function as direct defences [31]. We show that some Arabidopsis sRNA mutants display an altered susceptibility against *C. higginsianum*,

**Figure 1.** *Arabidopsis thaliana* leaves infected by *Colletotrichum higginsianum*, 6 days post inoculation.

**Figure 2.** *Zea mays* leaf (left) and root (right) infected with *Colletotrichum graminicola*.

together with a defective setup of chemical defences. Moreover, to better understand the role of sRNA during infection with *Colletotrichum* spp., we performed an miRNA expression pro‐ filing to obtain a deeper insight into adaptations of the sRNA transcriptome in different *C. graminicola*-infected maize tissues. The miRNA profiling demonstrated that the vast majority of altered miRNAs were targeting genes that are not directly linked to antifungal-defence pathways, suggesting that antifungal-defence responses are not regulated by specifically induced miRNAs.

This chapter provides a multi‐omics analysis of sRNA‐mediated antifungal plant reactions on a phenotypic, metabolomic as well as transcriptomic point of view. Altogether, our data pro‐ pose a rather indirect defensive role of sRNAs in calibrating metabolomic and transcriptomic balances during antifungal responses against *Colletotrichum* spp. Future putative applications of sRNA-based fungal control strategies will be commented.
