MicroRNA396-Targeted SHORT VEGETATIVE PHASE Is Required to Repress Flowering and Is Related to the Development of Abnormal Flower Symptoms by the Phyllody Symptoms1 Effector

Abstract

ORCID IDs: 0000-0002-9951-687X (C.-P.L.); 0000-0002-7295-5004 (S.-S.L.).Leafy flowers are the major symptoms of peanut witches' broom (PnWB) phytoplasma infection in Catharanthus roseus. The orthologs of the phyllody symptoms1 (PHYL1) effector of PnWB from other species of phytoplasma can trigger the proteasomal degradation of several MADS box transcription factors, resulting in leafy flower formation. In contrast, the flowering negative regulator gene SHORT VEGETATIVE PHASE (SVP) was up-regulated in PnWB-infected C. roseus plants, but most microRNA (miRNA) genes had repressed expression. Coincidentally, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the PHYL1 gene of PnWB (PHYL1 plants), which show leafy flower phenotypes, up-regulate SVP of Arabidopsis (AtSVP) but repress a putative regulatory miRNA of AtSVP, miR396. However, the mechanism by which PHYL1 regulates AtSVP and miR396 is unknown, and the evidence of miR396-mediated AtSVP degradation is lacking. Here, we show that miR396 triggers AtSVP messenger RNA (mRNA) decay using genetic approaches, a reporter assay, and high-throughput degradome profiles. Genetic evidence indicates that PHYL1 plants and atmir396a-1 mutants have higher AtSVP accumulation, whereas the transgenic plants overexpressing MIR396 display lower AtSVP expression. The reporter assay indicated that target-site mutation results in decreasing the miR396-mediated repression efficiency. Moreover, degradome profiles revealed that miR396 triggers AtSVP mRNA decay rather than miRNA-mediated cleavage, implying that AtSVP caused miR396-mediated translation inhibition. We hypothesize that PHYL1 directly or indirectly interferes with miR396-mediated AtSVP mRNA decay and synergizes with other effects (e.g. MADS box transcription factor degradation), resulting in abnormal flower formation. We anticipate our findings to be a starting point for studying the posttranscriptional regulation of PHYL1 effectors in symptom development.

Evolution of microRNA827 targeting in the plant kingdom

“…Frequently, the new miRNA-target modules are still involved in a similar regulation network as the original modules. The miR396 family is involved in controlling cell proliferation during leaf development by repressing several members in the GROWTH REGULATING FACTOR family and two other types of transcription factors identified recently in Arabidopsis thaliana and Catharanthus roseus (Liu et al, 2009;Rodriguez et al, 2009;Debernardi et al, 2012;Yang et al, 2015). Additionally, miR159 has been reported to have a conserved role in (Millar & Gubler, 2005;Buxdorf et al, 2010).…”

Structural insights into the interaction between phytoplasmal effector causing phyllody 1 and MADS transcription factors

“…Nicotiana benthamiana leaves (0.5 g harvested weight) were homogenized with 1 ml of immunoprecipitation buffer (125 m m TRIS‐HCl, pH 7.5, 150 m m NaCl, 1 m m EDTA, 5% glycerol) containing EDTA‐free Protease Inhibitor Cocktail (Roche, https://www.roche.com/). The homogenate was centrifuged at 14170 g for 10 min at 4°C to remove cell debris, and 0.5 ml of the supernatant was incubated with anti‐PHYL1 antibody (Yang et al ., ) and 25 μl of Protein A magnetic beads (GE Healthcare, https://www.gehealthcare.com/) at 4°C for 2 h. The beads were precipitated using a magnetic stand, and washed five times with 1 ml of wash buffer (125 m m TRIS‐HCl, pH 7.5, 150 m m NaCl, 1 mM EDTA, 5% glycerol, 0.1% TritonX‐100) for 5 min each at 4°C. Proteins were eluted from the beads by boiling with 2× SDS sample buffer (2% SDS, 1% β‐mercaptoethanol, 10% glycerol, 0.005% bromophenol blue, 50 m m TRIS base, pH 6.8).…”

“…Recently, the effectors that induce phyllody have been collectively labeled as phyllogens (Maejima et al, 2014). These phyllogens include the secreted aster yellows witches'-broom (AYWB) phytoplasma protein 54 (SAP54) and its close homolog, phytoplasmal effector causing phyllody symptoms 1, from onion yellows phytoplasma (PHYL1 OY ) and peanut witches'-broom phytoplasma (PHYL1 PnWB ) (MacLean et al, 2011;Maejima et al, 2014Maejima et al, , 2015Liu et al, 2015;Yang et al, 2015;Iwabuchi et al, 2019). The PHYL1 homologs PHYL1 OY and SAP54 have recently been shown to interact with Arabidopsis SEPALLATA1-4 (SEP1-4) and APETALA1 (AP1), both of which are MADS domain transcription factors that are assumed to be essential components of the floral quartets (MacLean et al, 2014;Maejima et al, 2014Maejima et al, , 2015.…”

Comparative Analysis of MicroRNA Expression in Three Paulownia Species with Phytoplasma Infection

“…miR5368g-5p was predicted to target ZEP, which is the key enzyme responsible for the accumulation of antheraxanthin and violaxanthin [66]. Yang, et al [67] reported that a high expression of ZEP increased the stored carotenoid pigments in transgenic tomato plants. In this study, ZEP was up-regulated in the three PaWB seedlings, implying that carotenoids were abundantly produced in phytoplasma-infected Paulownia leaves.…”