Pablo A. Manavella

Plant secondary siRNA production determined by microRNA-duplex structure

Processing of microRNA (miRNA) precursors results in the release of a double-stranded miRNA/miRNA* duplex. The miRNA or guide strand, is loaded onto the Argonaute (AGO) effector, and the miRNA* or passenger strand is typically degraded. The loaded AGO-containing RNA-induced silencing complex specifically recognizes a target mRNA, leading to its degradation or translational inhibition. In plants, miRNA-mediated cleavage of a target triggers in some cases the production of secondary small interfering RNAs (siRNAs), which in turn can silence other genes in trans. This alternative pathway depends on the length of the miRNA and the specific AGO in the effector complex. However, 22-nt miRNAs are sufficient, but not essential for this pathway. Using a combination of computational and experimental approaches, we show that transitivity can be triggered when the small RNA that is not retained in AGO is 22-nt long. Moreover, we demonstrate that asymmetrically positioned bulged bases in the miRNA:miRNA* duplex, regardless of miRNA or miRNA* length, are sufficient for the initiation of transitivity. We propose that the RNA-induced silencing complex reprogramming occurs during the early steps of miRNA loading, before the miRNA duplex is disassembled and the guide strand is selected.

Fast-Forward Genetics Identifies Plant CPL Phosphatases as Regulators of miRNA Processing Factor HYL1

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.

HAHB4, a sunflower HD-Zip protein, integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses

The Helianthus annuus (sunflower) HAHB4 transcription factor belongs to the HD-Zip family and its transcript levels are strongly induced when sunflower plants are attacked by herbivores, mechanically damaged or treated with methyl-jasmonic acid (MeJA) or ethylene (ET). Promoter fusion analysis, in Arabidopsis and in sunflower, demonstrated that induction of HAHB4 expression by these treatments is regulated at the transcriptional level. In transiently transformed sunflower plants HAHB4 expression upregulates the transcript levels of several genes involved in JA biosynthesis and defense-related processes such as the production of green leaf volatiles and trypsin protease inhibitors (TPI). In HAHB4 sunflower overexpressing tissue, increased activities of lipoxygenase, hydroperoxide lyase and TPI are detected whereas in HAHB4-silenced tissue these activities are reduced. Transgenic Arabidopsis thaliana and Zea mays plants ecotopically expressing HAHB4 also exhibit higher transcript levels of defense-related genes and when Spodoptera littoralis or Spodoptera frugiperda larvae are placed on each species, respectively, larvae consumed less and gain less mass compared with larvae feeding on control plants. Arabidopsis plants ectopically expressing HAHB4 had higher amounts of JA, JA-isoleucine and ET compared with control plants both before and after wounding, but reduced levels of salicylic acid (SA) after wounding and bacterial infection. We conclude that HAHB4 coordinates the production of phytohormones during biotic stress responses and mechanical damage, specifically by positively regulating JA and ET production and negatively regulating ET sensitivity and SA accumulation.

Transient transformation of sunflower leaf discs via an Agrobacterium -mediated method: applications for gene expression and silencing studies

The sunflower belongs to the Compositae family and is an economically important crop because of the quality of its oil. Unfortunately, molecular analyses are limited due to the lack of genomic information, mutant libraries and efficient and rapid transformation protocols. In a wide variety of species, Agrobacterium-mediated transient transformation is a useful tool that can provide valuable insight into many biological processes. However, this technology has not been routinely applied to the sunflower because of difficulties with infiltration. Here, we present an optimized protocol for Agrobacterium–mediated transient transformation of leaf discs. Using this procedure, we were able to quickly overexpress or silence a given gene, enabling us to study several biochemical processes and characterize sunflower regulatory sequences. One of the major advantages of this approach is that in only 1 work-week it is possible to acquire considerable molecular information while avoiding the use of controversial heterologous systems. Transforming heterologous species is frequently unacceptable, as the conservation of molecular events in many cases is not well documented.

Role of recently evolved miRNA regulation of sunflower HaWRKY6 in response to temperature damage

MicroRNAs (miRNAs) are small 21-nucleotide RNAs that post-transcriptionally regulate gene expression. MiR396 controls leaf development by targeting GRF and bHLH transcription factors in Arabidopsis. WRKY transcription factors, unique to plants, have been identified as mediating varied stress responses. The sunflower (Helianthus annuus) HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396. A possible post-transcriptional regulation of HaWRKY6 by miR396 was investigated. Here, we used expression analyses, performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and northern blots together with computational approaches to establish the regulatory interaction between HaWRKY6 and the identified sunflower miR396. Arabidopsis plants expressing a mi396-resistant version of HaWRKY6 confirmed the miRNA-dependency of the HaWRKY6 silencing. Sunflower plants exposed to high temperatures or salicylic acid presented opposite expression of HaWRKY6 and miR396. Experiments using the wildtype and miRNA-resistant versions of HaWRKY6 showed altered stress responses. Our results showed a role of the recently evolved miR396 regulation of HaWRKY6 during early responses to high temperature. Our study reveals how a miRNA that normally regulates development has been recruited for high-temperature protection in sunflower, a plant particularly well adapted to this type of stress.

HAHB10, a sunflower HD-Zip II transcription factor, participates in the induction of flowering and in the control of phytohormone-mediated responses to biotic stress

The transcription factor HAHB10 belongs to the sunflower (Helianthus annuus) HD-Zip II subfamily and it has been previously associated with the induction of flowering. In this study it is shown that HAHB10 is expressed in sunflower leaves throughout the vegetative stage and in stamens during the reproductive stage. In short-day inductive conditions the expression of this gene is induced in shoot apexes together with the expression of the flowering genes HAFT and HAAP1. Transgenic Arabidopsis plants expressing HAHB10 cDNA under regulation either by its own promoter or by cauliflower mosaic virus (CaMV) 35S exhibited an early flowering phenotype. This phenotype was completely reverted in a non-inductive light regime, indicating a photoperiod-dependent action for this transcription factor. Gene expression profiling of Arabidopsis plants constitutively expressing HAHB10 indicated that specific flowering transition genes such as FT, FUL, and SEP3 were induced several fold, whereas genes related to biotic stress responses, such as PR1, PR2, ICS1, AOC1, EDS5, and PDF1-2a, were repressed. The expression of HAHB10 and of the flowering genes HASEP3 and HAFT was up-regulated by both salicylic acid (SA) treatment and infection with a virulent strain of Pseudomonas syringae. Basal SA and jasmonic acid (JA) levels in Arabidopsis plants ectopically expressing HAHB10 were similar to those of control plants; however, SA levels differentially increased in the transgenic plants after wounding and infection with P. syringae while JA levels differentially decreased. Taken together, the results indicated that HAHB10 participates in two different processes in plants: the transition from the vegetative to the flowering stage via the induction of specific flowering transition genes and the accumulation of phytohormones upon biotic stresses.

miRNA Biogenesis: A Dynamic Pathway

MicroRNAs (miRNAs) modulate plant homeostasis through the inactivation of specific mRNAs, especially those encoding transcription factors. A delicate spatial/temporal balance between a miRNA and its targets is central to achieving the appropriate biological outcomes. In this review we discuss our growing understanding of the dynamic regulation of miRNA biogenesis. We put special emphasis on crosstalk between miRNA biogenesis and other cellular processes such as transcription and splicing. We also discuss how the pathway is regulated in specific tissues to achieve harmonious plant development through a subtle balance between gene expression and silencing.

Tissue-Specific Silencing of Arabidopsis SU(VAR)3-9 HOMOLOG8 by miR171a*

Summary: Active miR171a* complexes are common in Arabidopsis and trigger silencing of SUVH8, a new microRNA target that was acquired very recently in the Arabidopsis lineage. MicroRNAs (miRNAs) are produced from double-stranded precursors, from which a short duplex is excised. The strand of the duplex that remains more abundant is usually the active form, the miRNA, while steady-state levels of the other strand, the miRNA*, are generally lower. The executive engines of miRNA-directed gene silencing are RNA-induced silencing complexes (RISCs). During RISC maturation, the miRNA/miRNA* duplex associates with the catalytic subunit, an ARGONAUTE (AGO) protein. Subsequently, the guide strand, which directs gene silencing, is retained, while the passenger strand is degraded. Under certain circumstances, the miRNA*s can be retained as guide strands. miR170 and miR171 are prototypical miRNAs in Arabidopsis (Arabidopsis thaliana) with well-defined targets. We found that the corresponding star molecules, the sequence-identical miR170* and miR171a*, have several features of active miRNAs, such as sequence conservation and AGO1 association. We confirmed that active AGO1-miR171a* complexes are common in Arabidopsis and that they trigger silencing of SU(VAR)3-9 HOMOLOG8, a new miR171a* target that was acquired very recently in the Arabidopsis lineage. Our study demonstrates that each miR171a strand can be loaded onto RISC with separate regulatory outcomes.

KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1

Significance Micro RNAs (miRNAs) are small RNA molecules that regulate gene expression posttranscriptionally in a process known as gene silencing. Fine-tuning the production of miRNAs is essential for correct silencing of their targets, which in turn is important for homeostasis and development. To fine-tune the production of miRNAs, plants deploy a combination of proteins that act as cofactors of the miRNA-processing machinery. Here, we describe REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a tissue-specific regulator of miRNA biogenesis in plants. RCF3 interacts with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2), ultimately affecting the phosphorylation of one of the main DICER-LIKE1 (DCL1) accessory proteins, HYPONASTIC LEAVES1 (HYL1), with a concomitant effect on miRNA production. The biogenesis of microRNAs (miRNAs), which regulate mRNA abundance through posttranscriptional silencing, comprises multiple well-orchestrated processing steps. We have identified the Arabidopsis thaliana K homology (KH) domain protein REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a cofactor affecting miRNA biogenesis in specific plant tissues. MiRNA and miRNA-target levels were reduced in apex-enriched samples of rcf3 mutants, but not in other tissues. Mechanistically, RCF3 affects miRNA biogenesis through nuclear interactions with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2). These interactions are essential to regulate the phosphorylation status, and thus the activity, of the double-stranded RNA binding protein and DICER-LIKE1 (DCL1) cofactor HYPONASTIC LEAVES1 (HYL1).

Germline-transmitted genome editing in Arabidopsis thaliana Using TAL-effector-nucleases.

Transcription activator–like effector nucleases (TALENs) are custom-made bi-partite endonucleases that have recently been developed and applied for genome engineering in a wide variety of organisms. However, they have been only scarcely used in plants, especially for germline-modification. Here we report the efficient creation of small, germline-transmitted deletions in Arabidopsis thaliana via TALENs that were delivered by stably integrated transgenes. Using meristem specific promoters to drive expression of two TALEN arms directed at the CLV3 coding sequence, we observed very high phenotype frequencies in the T2 generation. In some instances, full CLV3 loss-of-function was already observed in the T1 generation, suggesting that transgenic delivery of TALENs can cause highly efficient genome modification. In contrast, constitutive TALEN expression in the shoot apical meristem (SAM) did not cause additional phenotypes and genome re-sequencing confirmed little off-target effects, demonstrating exquisite target specificity.