Wanhui Kim

Bacterial pathogens employ a large array of type IIIsecreted effectors to manipulate host cell immunity and metabolism. Ralstonia solanacearum species complex, the causal agent of bacterial wilt disease in numerous plant hosts, deploy a conserved subset of RipG effectors containing leucine-rich repeats and an F-box motif that collectively contribute to virulence and host specificity. RipG effectors are proposed to hijack the eukaryotic ubiquitin-proteasome machinery through the recruitment of substrates to host Skp1-cullin-F-box ubiquitinligase complexes via RipG F-box/host Skp1 adaptor interactions. However, only few host proteins have been reported to interact with RipG effectors. Here, using a surrogate type III delivery and a heterologous expression systems, we show that RipG6 can suppress plant pattern-triggered immunity in an F-box-dependent manner. We further identified a tomato receptor-like cytoplasmic kinase (SlRLCK-VIII-6) as an interactor of RipG6 in yeast and plant cells. SlRLCK-VIII-6 stability was reduced when co-expressed with RipG6 but not with the RipG6 variant lacking the F-box motif. Lastly, we provide evidence that Nicotiana benthamiana and Arabidopsis thaliana homologs of SlRLCK-VIII-6 can act as positive regulators of plant immune signaling. Together, our work supports a model where RipG6 destabilizes RLCK-VIII-6 possibly via its recruitment in a host ubiquitin-ligase complex in order to suppress plant immunity. Further research into RLCK-VIII role will enhance our understanding of the manipulation of plant immunity signaling by pathogen effectors.

Wanhui Kim, Hyelim Jeon, Hyeonjung Lee, Kee Hoon Sohn, and Cécile Segonzac. Mol. Cells 2023;46:710-24. https://doi.org/10.14348/molcells.2023.0127

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) threatens the cultivation of important crops worldwide. We sequenced 30 RSSC phylotype I ( R. pseudosolanacearum ) strains isolated from pepper (Capsicum annuum) and tomato (Solanum lycopersicum) across the Republic of Korea. These isolates span the diversity of phylotype I, have extensive effector repertoires and are subject to frequent recombination. Recombination hotspots among South Korean phylotype I isolates include multiple predicted contact-dependent inhibition loci, suggesting that microbial competition plays a significant role in Ralstonia evolution. Rapid diversification of secreted effectors presents challenges for the development of disease-resistant plant varieties. We identified potential targets for disease resistance breeding by testing for allele-specific host recognition of T3Es present among South Korean phyloype I isolates. The integration of pathogen population genomics and molecular plant pathology contributes to the development of location-specific disease control and development of plant cultivars with durable resistance to relevant threats.

Ralstonia solanacearum (Rso) is a causal agent of bacterial wilt in Solanaceae crops worldwide including Republic of Korea. Rso virulence predominantly relies on type III secreted effectors (T3Es). However, only a handful of Rso T3Es have been characterized. In this study, we investigated subcellular localization of and manipulation of plant immunity by 8 Rso T3Es predicted to harbor a nuclear localization signal (NLS). While 2 of these T3Es elicited cell death in both Nicotiana benthamiana and N. tabacum, only one was dependent on suppressor of G2 allele of skp1 (SGT1), a molecular chaperone of nucleotide-binding and leucine-rich repeat immune receptors. We also identified T3Es that differentially regulate flg22-induced reactive oxygen species production and gene expression. Interestingly, several of the NLS-containing T3Es translationally fused with yellow fluorescent protein accumulated in subcellular compartments other than the cell nucleus. Our findings bring new clues to decipher Rso T3E function in planta.

Binding of transcription factors to their target sequences is a primary step in the regulation of gene expression and largely determines gene regulatory networks. Chromatin immunoprecipitation (ChIP) is an indispensable tool used to investigate the binding of DNA-binding proteins (e.g., transcription factors) to their target sequences in vivo. ChIP assays require specific antibodies that recognize endogenous target transcription factors; however, in most cases, such specific antibodies are unavailable. To overcome this problem, many ChIP assays use transgenic plants that express epitope-tagged transcription factors and immunoprecipitate the protein with a tag-specific antibody. However, generating transgenic plants that stably express epitope-tagged proteins is difficult and time-consuming. Here, we present a rapid, efficient ChIP protocol using transient expression in Arabidopsis mesophyll protoplasts that can be completed in 4 days. We provide optimized experimental conditions, including the amount of transfected DNA and the number of protoplasts. We also show that the efficiency of our ChIP protocol using protoplasts is comparable to that obtained using transgenic Arabidopsis plants. We propose that our ChIP method can be used to analyze in vivo interactions between tissue-specific transcription factors and their target sequences, to test the effect of genotype on the binding of a transcription factor within a protein complex to its target sequences, and to measure temperature-dependent binding of a transcription factor to its target sequence. The rapid and simple nature of our ChIP assay using Arabidopsis mesophyll protoplasts facilitates the investigation of in vivo interactions between transcription factors and their target genes.

MicroRNAs originate from primary transcripts containing hairpin structures. The levels of mature miR156 influence the leaf number prior to flowering in the life cycle of plants. To understand the molecular mechanism of biogenesis of primary miR156a (pri-miR156a) to mature miR156, a base-pair opening dynamics study was performed using model RNAs mimicking the cleavage site of wild type and B5 bulge-stabilizing mutant pri-miR156a constructs. We also determined the mature miR156 levels and measured leaf numbers at flowering of plants overexpressing the wild type and mutant constructs. Our results suggest that the stabilities and/or opening dynamics of the C15·G98 and U16·A97 base-pairs at the cleavage site are essential for formation of the active conformation and for efficient processing of pri-miR156a, and that mutations of the B5 bulge can modulate mature miR156 levels as well as miR156-driven leaf number phenotypes via changes in the base-pair stability of the cleavage site.

Phanu Serivichyaswat, Hak-Seung Ryu, Wanhui Kim, Soonkap Kim, Kyung Sook Chung, Jae Joon Kim, and Ji Hoon Ahn. Mol. Cells 2015;38:259-66. https://doi.org/10.14348/molcells.2015.2311

The FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family proteins play an important role in the regulation of flowering time. In the Arabidopsis thaliana genome, there are six genes in the FT/TFL1 family. To determine how these FT/TFL1 family genes contribute to the regulation of flowering time, this study generated a comprehensive set of mutants (sixty-three multiple mutants in all combinations) of the FT/TFL1 family genes and analysed their flowering times at 23 and 16°C under long-day conditions. The analysis confirmed that FT and TFL1 are major determinants of flowering time under long-day conditions. At 23 °C, ft-10 tsf-1 mft-2 showed the latest flowering, whereas tfl1-20 atc-2 bft-2 showed the earliest flowering. Flowering occurred in the sextuple mutants. Introduction of tsf-1 led to reduced sensitivity to ambient temperature change. Introduction of tfl1-20 caused a stronger effect in accelerating flowering time at 16 °C than at 23 °C. Overexpression of miR156 did not block flowering of sextuple mutants, suggesting that there is a pathway to induce flowering independent of the FT/TFL1 pathway and miR156 pathway. This study proposes that this mutant population will be useful in further investigation of the functions of the FT/TFL1 family genes in plant development.

Plant microRNAs (miRNAs) are non‐coding RNAs that negatively regulate expression of their target genes. Although much is known about miRNA biogenesis and repression of target genes by miRNAs, the molecular mechanisms underlying the transcriptional regulation of miRNA itself are poorly understood. Here, we report that SHORT VEGETATIVE PHASE (SVP) protein is a direct transcriptional regulator of miR172. The levels of mature miR172 and pri‐miR172a were anti‐correlated with SVP activity. miR172a has multiple transcription start sites, among which the transcript starting with cytosine (−671 bp, relative to the mature miR172a) was a major species. EMSA and ChIP analysis demonstrated that SVP protein binds to the CArG motifs in the miR172a promoter. These results suggest that SVP protein directly regulates miR172 transcription in Arabidopsis.