Understanding the regulatory basis of defence signaling in plants : the role of Argonautes in modulating defense responses in Nicotiana attenuata

Plants display extensive transcriptional and phenotypic plasticity to adapt to unforeseen challenges and maximize their fitness. Such massive reprogramming requires an efficient reprogramming of gene expression in tissue and time-specific manner. At the same time, plants contain numerous non-coding small RNAs (smRNAs) such as microRNAs and small-interfering RNAs (siRNAs) that might generate an efficient and dynamic regulatory network during stress adaptation. An effective way to study ecological functions of smRNAs is to manipulate the expression of the genes responsible for their biosynthesis and action. The Argonaute (AGO) family of genes are amongst the core functional components of the machinery of the smRNA pathways as they form the RNA-induced silencing complex. As AGOs are the effectors of smRNA pathways and directly interact with smRNAs as well as their targets to modulate gene expression, they are one of the most important candidates for studying smRNA-mediated gene regulation. Here, we investigated the biological functions of the AGO’s in Nicotiana attenuata during its response to ecological challenges such as attack by insect herbivores and fungi. The N. attenuata genome encodes for 11 AGOs from 8 unique genes homologous to AGO1 (NaAGO1a, b, c), AGO2, AGO4 (NaAGO4a, b), AGO5, AGO7, AGO8, AGO9 and AGO10. In this investigation we determined which AGO(s) moderate the interaction of N. attenuata with its biotic challengers, namely insect herbivores, fungal pathogens and arbuscular mycorrhizal fungi (AMF). We performed loss-of-function characterization of the AGOs by stably silencing their expression. We discovered a high degree of specificity in response of AGOs to these diverse ecological interactions. AGO1 is an established regulator of plant development in Arabidopsis, it was also found to be essential in N. attenuata and stably silenced lines (homozygous with single insertions) could not be recovered. Our results show that AGO8 (along with AGO5) regulates plant direct defenses against herbivore attack, whereas only AGO4 is the regulator of signaling and resistance during infection with hemibiotrophic pathogenic fungi. Further, AGO7 is involved in installing optimum colonization of N. attenuata roots with mycorrhizal fungi and competitive fitness in resource limited environments. These conclusions are described in three manuscripts included in this thesis. Furthermore, our investigation hints at Summary 125 novel functions for AGO9 in impacting traits important to flower ecology and warrants further investigation. We have also described a large part of the non-coding smRNA fraction of N. attenuata’s transcriptome (smRNome), specially the miRNAs in the genome (miRNome). Using the high-throughput, next-generation sequencing (NGS, also often referred to as deep sequencing) approach of the Illumina-platform, we investigated the composition and temporal dynamics of expression of N. attenuata miRNome when the plants are subjected to herbivorous larvae of Manduca sexta and to the hemibiotrophic fungal pathogen, Fusarium brachygibbosum. On mapping the conservation across 34 families of plants, it was evident that nearly half of the miRNAs of N. attenuata miRNome are conserved. The patterns of accumulation of miRNAs were highly dependent on the type of stress (insect attack or fungal infection), and they were highly temporally dynamic in nature. A large fraction of these miRNAs was influenced by silencing specific AGOs such as AGO8 and AGO4 during response to insect and pathogen attack, respectively. Direct defenses on N. attenuata are under the control of the jasmonic acid (JA) signaling pathway, and a MYB-family transcription factor (MYB8) is a master regulator of biosynthesis of several key inducible defense metabolites. Therefore, we constructed networks of miRNA-modulated signaling and defense processes, such as the miRNA-MYB8 network for regulation of biosynthesis of direct defense metabolites during herbivore attack and the miRNA-mRNA network of JA-biogenesis and -signaling pathway during pathogen infection. Moreover, we validated the ecological functions of the AGOs by testing them in nature. The AGO4-silenced lines, the lines silenced in expression of other component of the smRNA biogenesis machinery, namely RNA-directed RNA polymerases 1 and 2 (RdR1, RdR2) and the Dicer-like protein (DCL3) as well as the binary combinations of AGO4 with RdR1/2 and DCL3 were released and evaluated under field conditions in the natural habitats of N. attenuata. We show the functional specificity of the smRNA machinery during hemibiotrophic pathogen interaction of the host. The pathway comprising the three core components of DCL3, RdR2/1 and AGO4 modulates disease resistance by regulating pathogen-induced JA-biogenesis and signaling. In conclusion, (i) the biological functions of the AGO family of genes was investigated during the plant response to three types of biotic interactions; a high level of specificity in AGO Summary 126 function was discovered. (ii) The miRNome and its stimulus-dependent reprograming to modulate defense signaling was uncovered. (iii) From our results, we infer that plants rely heavily on smRNAs to regulate signaling and defense responses in nature to circumvent stress and that a stress-dependent specialized smRNA-machinery is recruited for their action. The next step in this direction is to determine the function of other AGOs as well as to determine the biochemical mechanism underlying such eco-biological functions.