DICER‐like Proteins and Their Role in Plant‐herbivore Interactions in Nicotiana attenuata

Abstract

DICER-like (DCL) proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant-herbivore interactions remains unstudied. We identified and stably silenced four distinct DCL genes by RNAi in Nicotiana attenuata (Torrey ex. Watson), a model for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal. Manduca sexta larvae performed significantly better on ir-dcl3 and ir-dcl4 plants, but not on ir-dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir-dcl4 and double ir-dcl2 ir-dcl3 plants were impaired in JA accumulation, while JA-Ile was increased in ir-dcl3 plants. Ir-dcl3 and ir-dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir-dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti-herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions.

Identification and profiling of miRNAs during herbivory reveals jasmonate-dependent and -independent patterns of accumulation in Nicotiana attenuata

“…The targets of tasiRNA3, transcripts homologous to A. thaliana ARF2 , 3 and 4 were significantly reduced after W+OS elicitation (Figure  4), suggesting that Nat-miR390 could affect the architecture of roots and thereby regulate the production of nicotine, which is synthesized in the roots, or promote tolerance of herbivory by increasing mineral uptake or sugar storage in roots. Our previous study shows that silencing NaDCL4 , which in turn reduces the accumulation of tasiRNA3, dramatically impairs root growth and nicotine accumulation in N. attenuata [45]. We validated the microarray data by qPCR analysis ( Additional file 7), and the result showed similar expression patterns ( Additional file 8).…”

“…OS-elicitation dramatically changes the smRNA population in N. attenuata [42], and two major components of the smRNA pathway, RdRs and DCL proteins, function in biotic and abiotic stress responses [42-45]. Silencing of N. attenuata RdR1 , DCL3 , and DCL4 results in impaired defense responses against M. sexta herbivory [42,44,45].…”

“…Silencing of N. attenuata RdR1 , DCL3 , and DCL4 results in impaired defense responses against M. sexta herbivory [42,44,45]. Silencing either NaRdR1 or NaDCL4 impairs jasmonic acid (JA) accumulation, and co-silencing NaDCL3 and NaDCL4 reduces JA levels, indicating that RdR1/DCL4-mediated smRNAs are critical regulators of responses to insect herbivory.…”

Herbivore-Induced DNA Demethylation Changes Floral Signalling and Attractiveness to Pollinators in Brassica rapa

“…However, it remains challenging to establish a causative link between methylome changes and phenotypic responses since a) methylome structure is not completely independent from genetic variation [55], and b) other processes such as RNA interference may also induce phenotypic responses in plants [72]. Several studies have disentangled effects induced by methylome changes from other causes by either comparing methylomes of different organs within single plant individuals [34, 73], or introduction of DNA methylation changes with chemicals such as 5-azaC [36, 74, 75].…”

Global and local perturbation of the tomato microRNA pathway by atrans-activatedDICER-LIKE 1mutant

“…It also impedes the generation of dcl1 loss-of-function mutants by reverse genetics in plants whose transformation includes a regeneration phase in tissue culture. For example, attempts to stably silence Nicotiana attenuata DCL1 by RNAi have failed due to lethality of the regenerated transformants (Bozorov et al , 2012). …”