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A screen for proteins involved in parasitism from Meloidogyne hapla identifies an amphid localized transthyretin-like protein as a potential effector

Frederik Polzin
Doctoral thesis
Date of Examination: 2015-10-14
Date of issue: 2015-10-23
Advisor: Gleason, Cynthia Prof. Dr.
Referee: Gleason, Cynthia Prof. Dr.
Referee: Gatz, Christiane Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-9656-3

 

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Abstract

English

Root-knot nematodes (Meloidogyne sp.) present a constant threat to agriculture worldwide. When present in the field, they can infect roots and cause a distorted root structure. This strongly reduces crop production and cause crop losses worth billions of dollars annually. Due to recent banning of many front-line nematicides and the limited number of naturally of resistant plant cultivars, it is essential to understand how these pathogens are able to infect host plants and establish their feeding sites so that we can engineer novel resistance strategies. Nematodes can secrete proteins during the infection process to help them manipulate plant responses and establish feeding sites. We call these proteins “effectors.” Recent publications have shown that root-knot nematodes potentially secrete hundreds of effectors. Unfortunately, root-knot nematodes are a challenging pathogen to study, and all but a handful of root-knot nematode effectors have been characterized. To characterize novel nematode effectors, I employed a rapid screen that delivers effectors into the plant cell using the type three secretion system of Pseudomonas syringae pv. tomato DC3000. This screen was called the “effector detector vector” (EDV) screen. Seven effector candidates were identified from a bioinformatic search of the M. hapla proteome and were used in this EDV screen. Two candidates that could enhance bacterial growth on wild type Arabidopsis were prioritized for additional study. One candidate effector, called Mh270, became the focus of my thesis. Mh270 showed an increase in gene expression in the pre- and early stages of the nematode life cycle, which hinted at a role in parasitism. Mh270 encodes a transthyretin-like protein (TTR) whose transcript hybridized to chemosensory pores (the amphids) of the nematode. TTR proteins are often found the excretory/secretory products of parasitic nematodes, but their roles are unknown. Transgenic Arabidopsis lines ectopically expressing Mh270 were tested for altered nematode susceptibility and PAMP-triggered immunity (PTI). Several different elicitor-induced PTI responses were studied in the Mh270 transgenic plants (ROS burst, callose deposition, root growth inhibition, PTI-marker gene expression). The transgenic lines did not show altered susceptibility to root-knot nematode nor were they affected in any of the PTI responses that were tested. In addition, only when Mh270 was delivered by Pst DC3000 and not by less virulent Pseudomonas strains, which lacked certain effectors, could Mh270 enhance bacterial growth, suggesting Mh270 may work in conjunction with other proteins in the virulent bacteria in order to help enhance virulence. To gain even more information about the function of Mh270, the subcellular localization of Mh270 in plants was studied. A C-terminal GFP tagged Mh270 showed cytoplasmic localization in the roots while in the leaves, it showed chloroplastic localization. Meanwhile, a yeast-two-hybrid screen revealed that Mh270 can interact with a mitochondrial membrane-localized Arabidopsis thaliana voltage dependent anion channel (AtVDAC3) in yeast. Possible explanations regarding Mh270’s differential in localization in roots and leaves and in regards to its interaction with an AtVDAC-partner will be discussed.
Keywords: Meloidogyne; effector
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