split-green-fluorescent-protein-system-to-visualize-effectors

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Seoul National University

The very late-flowering behavior of Arabidopsis winter-annual ecotypes is conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). A MADS-domain gene, AGAMOUS-LIKE 20 (AGL20), was identified as a dominant FRI suppressor in activation tagging mutagenesis. Overexpression of AGL20 suppresses not only the late flowering of plants that have functional FRI and FLC alleles but also the delayed phase transitions during the vegetative stages of plant development. Interestingly, AGL20 expression is positively regulated not only by the redundant vernalization and autonomous pathways of flowering but also by the photoperiod pathway. Our results indicate that AGL20 is an important integrator of three pathways controlling flowering in Arabidopsis.

The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis.

Autophagy is important for degradation and recycling of intracellular components. In a diversity of genera and species, orthologs and paralogs of the yeast Atg4 and Atg8 proteins are crucial in the biogenesis of double-membrane autophagosomes that carry the cellular cargoes to vacuoles and lysosomes. Although many plant genome sequences are available, the ATG4 and ATG8 sequence analysis is limited to some model plants. We identified 28 ATG4 and 116 ATG8 genes from the available 18 different plant genome sequences. Gene structures and protein domain sequences of ATG4 and ATG8 are conserved in plant lineages. Phylogenetic analyses classified ATG8s into 3 subgroups suggesting divergence from the common ancestor. The ATG8 expansion in plants might be attributed to whole genome duplication, segmental and dispersed duplication, and purifying selection. Our results revealed that the yeast Atg4 processes Arabidopsis ATG8 but not human LC3A (HsLC3A). In contrast, HsATG4B can process yeast and plant ATG8s in vitro but yeast and plant ATG4s cannot process HsLC3A. Interestingly, in Nicotiana benthamiana plants the yeast Atg8 is processed compared to HsLC3A. However, HsLC3A is processed when coexpressed with HsATG4B in plants. Molecular modeling indicates that lack of processing of HsLC3A by plant and yeast ATG4 is not due to lack of interaction with HsLC3A. Our in-depth analyses of ATG4 and ATG8 in the plant lineage combined with results of cross-kingdom ATG8 processing by ATG4 further support the evolutionarily conserved maturation of ATG8. Broad ATG8 processing by HsATG4B and lack of processing of HsLC3A by yeast and plant ATG4s suggest that the cross-kingdom ATG8 processing is determined by ATG8 sequence rather than ATG4.

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4.

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFP) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10), the two proteins reconstitute GFP fluorescence. We generated a number of  transgenic lines expressing sfGFP1-10 targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using  effectors AvrB and AvrRps4 in  and transgenic Arabidopsis plants. The versatile split sfGFP system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.

Spatiotemporal Monitoring of  Effectors via Type III Secretion Using Split Fluorescent Protein Fragments.

The pepper Pvr4 encoding coiled-coil (CC) nucleotide-binding (NB) leucine-rich repeat (LRR) protein (CC-NLR) confer hypersensitive resistance (HR) to potyviruses, including Pepper mottle virus (PepMoV) by recognizing the viral avirulence protein, NIb. To figure out the Pvr4-mediated HR mechanism, we analyzed the signaling component genes and structure-function relationships of Pvr4 using chimeras and deletion mutants in Nicotiana benthamiana. Molecular chaperone components including HSP90, SGT1 and RAR1 were required, while plant hormones and mitogen-activated protein (MAP) kinase signaling components had little effect on Pvr4-NIb-mediated HR cell death. Domain swap analyses indicated that the LRR domain of Pvr4 determines recognition of PepMoV-NIb. Our deletion analysis further revealed that CC domain or CC-NBARC domain alone can trigger autoactive cell death in N. benthamiana. However, the fragments having only LRR domain could suppress CC-NBARC domain-induced cell death in trans. Further, C-terminal truncation analysis of Pvr4 revealed that minimum three out of five LRR exons showing high similarity was essential for Pvr4 function. The LRR domain may maintain Pvr4 in an inactive state in the absence of NIb. These results provide further insight into the structure and function of NLR protein signaling in plants.

Article	Year
The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes & development\| PubMed ID: 10995392	2000
Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4. Autophagy\| PubMed ID: 27540766	2016
Spatiotemporal Monitoring of Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. The Plant cell\| PubMed ID: 28619883	2017
Coiled-coil and leucine-rich repeat domain of the potyvirus resistance protein, Pvr4, has distinct role in signaling and pathogen recognition. Molecular plant-microbe interactions : MPMI\| PubMed ID: 29663867	2018

Eunsook Park

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