MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs

Abstract

Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) ''phased'' intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all~540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent ''two-hit'' phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

On plant defense signaling networks and early land plant evolution

“…At the level of expression, they can be regulated by transcriptional as well as post-transcriptional means [95]. The latter is mediated by microRNAs (miRNAs) in angiosperms [96-99]. Several NBS-LRR -targeting miRNA families exist, but due to the broad distribution of the miR482/2118 family, this family has received more attention than others.…”

“…The family first emerged in gymnosperms [96,100], which seems to coincide with an expansion of NBS-LRRs during this time period [88]. The coniferous plant P. abies has one of the largest expansions of miR482/2118 [8,100] and the genes likely originated through inverted duplication of NBS-LRR genes [100].…”

Plant MicroRNAs Display Differential 3' Truncation and Tailing Modifications That Are ARGONAUTE1 Dependent and Conserved Across Species

“…Given that most Arabidopsis miRNAs are 21 nucleotides in the wild-type background, it is possible that some or many miRNA targets produce tasiRNAs in the hen1 mutant because the miRNA variants that are tailed to 22 nucleotides. We performed a genome-wide analysis of sRNA phasing (Zhai et al, 2011) with the hen1 mutant sRNA libraries. Only the two loci AT2G45160 and AT3G60630, both of which regulate the development of shoot meristem (Schulze et al, 2010), produced phased siRNAs in hen1 mutants but not the wild type ( Figure 4A; see Supplemental Figure 5 online).…”

“…The head portion of reads was also mapped to the annotated transposable elements in TAIR (version 10; used only for its repeat data) to filter the transposable element-associated sRNAs used in Supplemental Figure 7 online, which were mostly ;24 nucleotides in the wild-type libraries. Genome-wide phasing analysis of sRNAs was performed as described previously (Zhai et al, 2011).…”

Deep Sequencing from hen1 Mutants to Identify Small RNA 3' Modifications

“…These double-stranded RNA (dsRNA) precursors are processed by DCL proteins into multiple distinct siRNA classes that represent both strands of the original dsRNA (Baulcombe 2004;Chapman and Carrington 2007). Plant siRNAs come in several forms; for example, heterochromatic siRNAs are produced from RNA polymerase IVdependent precursors generated from a wide variety of repetitive sequences (Wierzbicki et al 2008), whereas trans-acting or phased siRNAs are produced via Pol II from long, noncoding mRNAs or even from protein-coding transcripts (Montgomery et al 2008;Cuperus et al 2010;Zhai et al 2011). The Arabidopsis genome includes four DCL genes that are responsible for generating distinct classes of sRNAs and of different sizes, ranging from 21 to 24 nucleotides.…”