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Functional Analysis of Meloidogyne Graminicola C-Type Lectins and Their Role in the Nematode – Rice Interaction

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Functional Analysis of Meloidogyne Graminicola C-Type Lectins and Their Role in the Nematode – Rice Interaction University of Ghent Faculty of Science Department of Biology Academic year 2013 – 2015 Functional Analysis of Meloidogyne graminicola C-type Lectins and their Role in the Nematode – Rice Interaction Romnick Latina Promotor: Prof. Dr. Godelieve Gheysen Thesis submitted to obtain the degree Supervisor: Silke Nowak of Master of Science in Nematology Functional analysis of Meloidogyne graminicola C-type lectins and their role in the nematode – rice interaction Romnick LATINA Faculty of Science, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium Summary – Due to rice cropping intensification and increasing water scarcity, Meloidogyne graminicola has become a threat to rice production. With the aid of molecular tools and techniques, the host-nematode interaction has long been studied in the hope of finding control measures for this ominous rice root-knot nematode. In many investigations, proteins termed as effectors have been shown to mediate mechanisms and processes essential for pathogenesis of plant parasitic nematodes. Many of these effectors have been identified to unravel their functional roles. In this present work, two putative effector genes (UK41 and UK42) coding C-type lectins were investigated to know their roles in the M. graminicola - rice interaction. To gain some insights on their action site, eGFP fusion constructs were transiently expressed in N. benthamiana. Subcellular localizations revealed that UK41 localized to both the cytoplasm and nucleus while UK42 showed strong nuclear localization. To check their effect on host defenses, ETI and PTI assays were performed using transient expression in tobacco plants. Based on the R/Avr-gene pairs tested, both C-type lectins were found to be non-suppressive of the ETI. Transient assay using the PAMP, INF1, also indicated non-suppression of PTI. However, ROS production as a result of flg22- triggered PTI was found to be affected by both lectin genes. The oxidative burst in tobacco leaves was found to be delayed in the presence of both C-type lectins. Since there were some variabilities using the parameter maximum peak or oxidative burst, the new parameter, rate of reaction, was utilized. This parameter revealed that UK41 and UK42-treated leaves had lower reaction rates similar to MP10-treated leaves reflective of suppressed ROS production. These results suggest a role for C-type lectins in the M. graminicola and rice interaction. For future research, the nematode effector genes were transformed into rice and in the course of my MSc thesis, the transformed lines were studied for the presence and expression of the transgenes. Differential expression was found in both UK41 and UK42 transformed rice plants although in general, UK42 transformants had higher gene expression. Keywords – M. graminicola, C-type lectins, confocal microscopy, ROS assay, oxidative burst, flg22, qPCR, effector 2 The rice root-knot nematode, Meloidogyne graminicola, has been one of the most economically and globally important plant-parasitic nematodes. This species was first described in 1965 by Golden and Birchfield from grasses and oats in Louisiana. Since then, it has been found infecting primarily upland, irrigated and deep-water rice planted in South and Southeast Asia, China, South Africa, United States of America, Colombia and Brazil. M. graminicola is well- adapted to flooded conditions and can survive as eggs in egg masses or as juveniles in waterlogged conditions for a long time, particularly in rice remnants and it can remain viable for 14 months. It prefers 20-30% soil moisture, and thrives in dry soils during the tillering and panicle initiation of the rice plant (Dutta et al., 2012; Prot & Matias, 1995). In India, this nematode is more prevalent in dry nursery beds than in wet beds (Dongre & Simon, 2013) while in Myanmar, the population density of second stage juveniles in irrigated lowland fields is higher during summer compared to the rainfed season (Win et al., 2013). Meloidogyne graminicola has a very short life cycle that can be completed in 15 days at 27-37°C (Jaiswal & Singh, 2010). The swollen female lays her eggs in masses within the root cortex and juveniles hatch from the eggs. The infective juveniles (J2) remain in the maternal gall or migrate in the same root allowing them to multiply even under flooded conditions. In flooded conditions, these nematodes cannot invade rice plants but they can quickly resume their invasion when fields are drained by attacking behind the root tip. They can also thrive in some common rice field weeds such as Echinocloa, Cyperus and Panicum (Bridge & Starr, 2007). They use their stylet to penetrate the root cells, inject secretions and extract cell nutrients. After penetration inside the roots, they migrate in between the cortex cells to the region of cell differentiation. This intercellular movement causes the separation of the cells along the middle lamella. The J2 nematodes move towards the root tip and turn around when they reach the apical meristems to overcome the barrier formed by the endodermis and move back to the zone of differentiation. At this zone, they become sessile and start to swell with their head feeding on the protoxylem and protophloem cells. This is also where they induce the development of giant cells as part of their permanent feeding site (Karssen et al., 2013). In recent years, the complex interaction of plant-parasitic nematodes including M. graminicola with their host plants has been scrutinized. Since the discovery of the first ‘parasitism gene’ isolated from Globodera rostochiensis and Heterodera glycines and encoding β-1,4- 3 endoglucanase that degrades cellulose in plant cell walls during migration (Smant et al., 1998), other genes encoding for proteins termed as ‘effectors’ that are involved in nematode parasitism have been unearthed and studied (Haegeman et al, 2013). Most of these effectors have roles in migration and penetration such as expansins, (Qin et al., 2004), arabinogalactan endo-1,4-beta- galactosidase (Vanholme et al., 2009) and pectate lyase (Kudla et al., 2007), plant defense suppression such as SPRYSECs from G. pallida (secreted SPla and the RYanodine Receptor- containing proteins) (Postma et al., 2012) and Hs10A06 from H. schachtii (Hewezi et al., 2010), nematode protection like the GSTs or glutathione-S-transferases from M. incognita (Dubreuil et al., 2007) and feeding site maintenance and induction such as the 19CO7 from H. schachtii (Lee et al., 2011) and Hg-eng-1 from H. glycines (Bakhetia et al., 2007). In the M. graminicola J2 transcriptome, Haegeman et al. (2013) identified several putative effectors that are functional during early parasitism. The presence of signal peptides combined with the absence of a transmembrane domain is an identifier of a secreted protein and was considered in the analysis. These putative efffectors include homologues of plant cell wall- modifying enzymes such as expansins and pectate lyase, secreted proteins for detoxification, fatty acid and retinol-binding protein, annexin, calreticulin, chitinase, transthyretin-like protein, mitogen-activated protein (MAP-1), galectin, C-type lectins (CTLs) etc. In-situ hybridization of these putative effectors revealed their expression in the amphids, dorsal glands and subventral glands. Among these putative effector proteins, C-type lectins have been found abundantly not just in M. graminicola but in other nematodes as well. These types of lectins are calcium-dependent and previously known to be just carbohydrate-binding animal proteins. However, evolutionary studies have revealed that many CTL domains (CTLDs) have evolved to specifically recognize protein, lipid and inorganic ligands as well (Zelensky & Gready, 2005). Generally, CTLDS function in adhesion and pathogen recognition and phagocytosis (Cambi & Figdor, 2003). In the genome of the model organism, C. elegans, 278 CTLDs containing proteins were found. Some of these genes (61 genes or 22% of all CTLD proteins) are found to be upregulated in the presence of a pathogen and 45 of these have a signal sequence. These secreted pathogen-induced proteins were proposed to contribute not only to pathogen recognition but are also involved in antigen- antibody interaction or possess antimicrobial activity (Schulenberg et al., 2008). CTLs have also been found in the gastrointestinal nematode parasites Heligmosomoides polygyrus, 4 Nippostrongylus brasiliensis (Harcus et al., 2009), Ancylostoma ceylanicum (Brown et al., 2007) and Toxocara canis (Loukas et al., 1999). In M. chitwoodi, the nematode secretome was found to have a CTL domain which is homologous to CTLs found in snake venoms (Roze et al., 2008). Very recent studies demonstrated that also in the reniform nematode, Rotylenchulus reniformis, the transcriptome of parasitic females has sequences homologous to CTLs (Ganji et al., 2014) and in G. pallida, the transcriptome of J2 nematodes contains CTL domains (Cotton et al., 2014). Even though it has always been implicated that CTLs play an important role in the immune system and parasitism of nematodes, there is no direct evidence yet on their function in plant- nematode interactions. The fact that these CTLs in M. graminicola contain signal peptides without a transmembrane domain, are found in the subventral glands of J2 nematodes and are upregulated in J2, is very suggestive of their involvement in nematode parasitism. Hence, this study was designed to identify the function of M. graminicola C-type lectins in rice-nematode interaction
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