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Functional characterization of phytoplasma candidate virulence proteins that target plant cell nuclei

Lead Research Organisation: John Innes Centre
Department Name: UNLISTED

Abstract

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Technical Summary

Phytoplasmas are insect-transmitted intracellular bacterial pathogens that infect plants in domesticated (crops) and natural habitats worldwide. Typically, they induce shoot proliferation and differentiation of floral parts into leafy structures, suggesting interference with basic cellular and developmental pathways. The applicant has led a consortium that sequenced the 724 kbp genome of aster yellows phytoplasma strain witches' broom (AY-WB), and is involved in two further phytoplasma sequencing programmes. Analysis of the AY-WB sequence identified 56 candidate virulence proteins of which 4 have nuclear localization signals (NLSs). Two of these proteins (A11 and A30) target nuclei in plant cells. The objective of this proposal is to functionally characterize those candidate virulence proteins that target plant nuclei, with the hypothesis that they could change gene expression profiles through interactions with transcription factors and regulatory sequences. This work takes advantage of the broad host range of AY-WB that includes Nicotiana benthamiana, tomato, and Arabidopsis thaliana. The specific aims of the proposal are: 1. Determine the cellular localization of all AY-WB NLS-containing proteins in plants. 2. Assess whether A11 and A30 differentially regulate transcription factors and how this may affect host susceptibility to AY-WB. Relative to other work on bacterial virulence in plants, the work is distinctive in focusing on virulence factors that target nuclei and on intracellular bacterial pathogens. Finally, this project will transfer research experience and knowledge obtained during a approximately 7 year career in the USA to the John Innes Centre, Norwich, UK.

Planned Impact

unavailable
 
Description We previously sequenced the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) and developed a bioinformatics pipeline to identify proteins that are secreted by this bacterium. We identified 56 candidates, which were named secreted AY-WB proteins (SAPs). We hypothesized that the 56 SAPs are candidate virulence proteins (effectors) that modulate processes of the AY-WB plant host and insect vector.

We found that phytoplasma effector proteins SAP11 and SAP30 accumulate in plant cell nuclei, whereas 6 other secreted AY-WB proteins (SAPs) did not. SAP30 locates specifically to nucleoli of plant cells. The localization of SAP30 in the nucleolus was confirmed by colocalization of SAP30 with fluorescently tagged Arabidopsis thaliana fibrillarin 1, which is known to accumulate in plant cell nucleoli.

Production of SAP11 in A. thaliana leads to the differential regulation of plant developmental genes and downregulation of genes involved in leaf senescence and secondary metabolism. These plants have large kale-like crinkled leaves, delayed flowering and produce more young shoots. The gene transcription profiles and phenotype of Arabidopsis plants that manufacture SAP11 overlap with those of AY-WB-infected plants and plant lines in which the production of the transcription factors is inhibited. We envision that the SAP11-mediated delay of leaf senescence and increase in shoots and leaf surface enhances and prolong opportunities for AY-WB replication and feeding and egg laying of AY-WB insect vectors. Thus, SAP11 has a true effector function.

We demonstrated that the nuclear localization of SAP11 is essential for the induction of the leaf serration phenotype.

35S:SAP30 transgenic A. thaliana lines did not show obvious phenotypic changes.

We generated antibodies to the SAP11 protein and used this antibody to investigate if we can detect SAP11 in the insect vector. We found that SAP11 is abundantly present in the insect salivary gland, particularly in the canaliculi that collect the salivary proteins for secretion into plants during insect feeding. Thus, insects may introduce SAP11 into plant cells simultaneously with the phytoplasmas.

We conducted plant transcript expression profiling at two stages of infection of phytoplasma-infected A. thaliana plants. Whereas at an early stage of infection only few A. thaliana genes were differentially regulated, a week later more plant genes were differentially regulated. This included numerous genes involved in plant development, such as MADS-box transcription factors. These results indicated that AY-WB interferes with plant development consistent with the phenotypic changes observed in AY-WB-infected Arabidopsis plants.

Comparison of the expression profiles of phytoplasma-infected and 35S:SAP11 transgenic A. thaliana lines identified several plants genes in common. These included three transcription factors and other genes involved in plant development. We also found that one third of the differentially regulated genes in 35S:SAP11 plants overlapped with those of the published miR319/JAW overexpression lines confirming later observations (BBSRC grant BB/G001928/1) that SAP11 destabilizes class II TCP transcription factors. The differential regulation of the plant genes is co-related with nuclear targeting and expression levels of SAP11 and SAP11 derivatives.
Exploitation Route Results generated in this project lead to additional project funding, including BB/G001928/1.
Sectors Agriculture

Food and Drink

Chemicals

Education

Environment
URL http://www.jic.ac.uk/staff/saskia-hogenhout/Phytoplasma/index.html
 
Description Phytoplasmas are insect-transmitted plant pathogens that can modulate their plant and insect hosts for their own benefit. Indeed, phytoplasmas interfere with plant development often leading to the production of more young leaf-like tissues and inhibition of flowering and plant senescence. This provides more leaf tissue for phytoplasma replication and better opportunities for feeding and egg laying of the phytoplasma insect vectors, which disperse phytoplasmas to new plant hosts. This project led to a better understanding of phytoplasma interactions with their plant hosts and insect vectors. This is important, because these pathogens are becoming increasingly a problem in (durable) crops, flower bulbs and ornamentals worldwide, including third world countries. This project impacted society at several levels: (A) Interactions among science disciplines; (B) Interaction with industry; (C) Training of young scientists; (D) Communication of science to (inter)national science communities; and (E) Engagement with the general public. (A) Interactions among science disciplines This research is interdisciplinary, as it unified the study of organisms spanning three Kingdoms (namely Prokaryota, Animalia, and Plantae) and therefore incorporated microbiology, entomology, and plant biology. (B) Interaction with industry Seed companies and experimental research stations frequently requested information about phytoplasmas, as they were affecting various crops, flower bulbs and ornamentals in northern Europe. Visited and presented talks at agrochemical and seed breeding companies: (C) Training of young scientists This funding contributed to start of the research program of S. Hogenhout, who just had moved her research program from the USA to the JIC, UK. The funding was used to generate preliminary data for a BBSRC grant proposal, which got funded (BB/G001928/1). (D) Communication of science to (inter)national science communities S. Hogenhout was invited to present talks at the following international meetings and research centers: - International Congress Molecular Plant-Microbe Interactions, Quebec City, Canada, July, 2009; - RIKEN Plant Science Centre, Yokohama, Japan, Jan, 2009; - Iwate Biotechnology Research Centre, Kitakami, Japan, Jan, 2009; - Max Planck Institute for terrestrial Microbiology, Marburg, Germany, Oct, 2008; - Max Planck Institute for Plant Breeding Research, Cologne, Germany, May, 2008; - Citrus Research and Extension Center (CREC), Lake Alfred, FL, USA, April, 2008; - Keystone Plant Innate Immunity conference, Keystone, Colorado, USA, Feb, 2008; - First International Phytoplasmologist Working Group Meeting, Bologna, Italy, Nov 2007; - International Congress on Molecular Plant-Microbe Interactions, Sorrento, Italy, July, 2007. S. Hogenhout was invited to present talks at the following national meetings and research centers: - The Scottish Crop Research Institute (SCRI), Dundee, Scotland, Sept, 2009; - Faculty of Life Sciences, University of Manchester, April, 2009; - Annual Science Meeting, Norwich, Oct, 2008; - Rothamsted Research Park, Harpenden, Nov, 2008; - University of Birmingham, School of Biosciences, Edgbaston, July, 2008; - Norwich Research Park Meeting "Microbes in Norwich", Norwich, April, 2008; - University of East Anglia, Norwich, Jan, 2008; - University of Nottingham, Division of Plant and Crop Sciences, Sutton Bonington, Nov, 2007; - XVI International Plant Protection Congress, Glasgow, Scotland, Oct, 2007; - British Society of Plant Pathology (BSPP) meeting, University of Bath, Sep, 2007. (E) Engagement with the general public I was a panel member of working group "Identifying Priorities for Research: Citrus Greening Meeting of Experts" organized by the NRC and Florida Department of Citrus, Ft Pierce and West Palm Beach, Florida, USA. 2007. Meetings involved discussions with farmers and owners of citrus groves.
First Year Of Impact 2008
Sector Agriculture, Food and Drink,Education,Environment
Impact Types Cultural

Societal