Expansins are among plant cell wall modifying agents specifically expressed during development of nematode-induced syncytia

Abstract

Cyst nematodes are economically important pests. As obligatory biotrophic endoparasites they invade host roots and induce formation of syncytia, structures that serve them as the only source of nutrients. During syncytium development, extensive cell wall modifications take place. Cell wall dissolution occurs during cell wall opening formation, cell walls expand during hypertrophy of syncytial elements and local cell wall synthesis leads to the thickening of syncytial cell wall and the formation of cell wall ingrowths. Numerous studies revealed that nematodes change expression of plant genes encoding cell wall modifying proteins including expansins. Expansins poses unique abilities to induce cell wall extension in acidic pH. Recently, we demonstrated that two α-expansin genes LeEXPA4 and LeEXPA5 are upregulated in tomato roots infected with potato cyst nematode (Globodera rostochiensis). In this addendum, we present the most recent results concerning involvement of plant cell wall modifying genes in syncytium development and discuss possible practical applications of this knowledge for developing plants with resistance against nematodes.

Plant parasitic nematodes cause substantial loses to crop plants.¹ Cyst nematodes invade the roots of hosts in their elongation and differentiation zones and migrate intracellularily searching for cells which properly react to their activity. These initial syncytial cells (ISC) are the starting points for the development of the specific feeding site, called syncytium. It is believed, that syncytia are induced by nematode secretions that interfere with the host cell thus leads to profound changes in the root morphogenetic program.²

During nematode infection plant cell wall undergoes extensive modifications. At the beginning of the interaction, once the nematode enters the root, it is degraded to enable migration and selection of the ISC. To achieve this, nematode uses its stylet and its own set of cell wall modifying proteins. Endo-β-1,4-glucanases,^3,4 pectate lyases⁵ and expansin-like compounds^6,7 were identified among nematode genes involved in wall degradation during migration phase. The process of syncytium development itself is also accompanied by cell wall re-modelling, including locally and temporarily regulated processes of synthesis and disassembly of cell wall polymers.⁸ The cell wall is locally dissolved to create openings between the ISC and neighbouring cells (Fig. 1A). As a result protoplasts of adjacent cells fuse and multinucleate syncytia are formed. Once a cell is incorporated into the syncytium it differentiates into a syncytial element: it becomes hypertrophied and requires extensive cell wall loosening and expansion (Fig. 1B). In addition, to enable proper functioning of the nematode feeding site the outer syncytial wall thickens to counteract the high osmotic pressure⁹ and cell wall ingrowths develop at the vessel-syncytium interface to increase efficiency of short-distance transport. To carry out all this processes parasite triggers host cell wall modifying proteins, such as endo-β-1,4-glucanases,^10,11 polygalacturonases¹² and expansins.¹³

Figure 1.

The ultrastructure and anatomy of tomato root infected with potato cyst nematode (G. rostochiensis). (A) a cell wall opening (arrow) formed in cell wall (CW) of 1 day old syncytium. (B) cross section of the susceptible tomato root containing 10 day old syncytium (asterisks). (C) cross section of transgenic tomato root with silenced expression of LeEXPA5 and containing 10 day old syncytium (asterisks). Abbreviations: GA, Golgi apparatus; N, nematode; Nu, nucleus; Pl, plastid. Scale bars: (A) 1 µm, (B and C) 50 µm.

Expansins are extracellular proteins and are regarded to be responsible for cell wall extension in acidic pH.¹⁴ In Arabidopsis thaliana roots infected with Heterodera schachtii at least ten expansin genes are expressed and two expansin genes are silenced.¹³ Differentially expressed expansin genes show temporarily and spatially regulated specific expression patterns. In a recent paper we investigated the expression patterns of 10 known α-expansin genes in tomato roots infected with potato cyst nematode (Globodera rostochiensis).¹⁵ We demonstrated that six tomato expansin genes (LeEXPA1, -2, -4, -5, -11 and -18) are upregulated upon nematode infection. Using in situ methods we localized transcripts of LeEXPA4 and -5 in different types of cells: LeEXPA4 expression was mainly localized in vascular cylinder cells located next to growing syncytia, while LeEXPA5 expression was localized in hypertrophying syncytial elements. The results obtained using immunogold localization method excluded the hypothetical involvement of expansin 5 in cell wall opening formation and provided evidence that this protein can act as a cell wall loosening agent during cell wall extension in enlarging syncytial elements.¹⁵

The involvement of expansins in nematode feeding site development seems to be a common event, regardless of the type of host-parasite combination. Recent microarray experiments revealed that expansin genes: EXPL2 and EXPR3 are specifically upregulated in soybean roots infected with soybean cyst nematode (Heterodera glycines).^16,17 Moreover, expansins were found to be specifically regulated during development of giant cells induced by root-knot nematodes, yet another group of sedentary endoparasitic nematodes.^18,19

Regarding the cell wall complexity and dynamics²⁰ it is highly probably that a set of different plant wall modifying agents is required to mediate process of cell wall expansion in enlarging syncytial elements. There are many evidences that genes encoding cellulases play a role in remodelling of syncytial cell walls.^10,11,21 In tomato, two genes encoding Sl-cel7 and Sl-cel9C1 endoglucanases are recruited by potato cyst nematode for feeding site induction.²¹ Interestingly, transcripts of cel7 and LeEXPA5 show overlapping expression patterns in developing syncytia.^15,21 The expression of cel7 similarly to LeEXPA5 was specifically induced in syncytial elements during early stages of syncytium development (1–5 dpi) and, at later developmental stages (7–10 dpi), it was limited to the cells recently incorporated into syncytium. Thus, it seems that LeEXPA5 and cel7 play synergistic or complementary roles in the cell wall restructuring during expansion of syncytial elements. Expansins might disrupt non-covalent bonds between cellulose microfibrills and hemicelluloses thus exposing xyloglucan and non-crystalline cellulose for hydrolysis by endoglucanases. Rapid cell growth accompanying enlargement of walls is a common event in plant development, e.g., in growing tomato fruit. Interestingly, parallel expression of LeEXPA5 and cel7 was detected also in tomato fruit at immature green and maturing green stages.^22,23 Thus, cell wall remodelling in hypertrophying syncytial elements and cell wall modification in rapidly growing fruit cells seems to be mediated with the same set of proteins belonging to the endoglucanase and expansin gene families. It is likely, that other proteins facilitating wall modification like xyloglucan endotransglycosylases (XET) are involved in the changes of syncytial walls, as mRNA of these genes was localized in 5 days old syncytia during soybean H. glycines interaction.²⁴ XETs catalyze endo-cleavage of xyloglucan polymers and transfer of newly generated reducing ends to other xyloglucan.^25,26 Therefore, it is tempting to speculate that another gene(s) expressed during tomato fruit expansion and representing xyloglucan endotransglycosylase family is(are) involved in hypertrophy of syncytial elements.

Little is known about the regulation of expression of plant genes in nematode infected roots. According to a generally accepted hypothesis, there are two possible ways of specific activation of plant genes in nematode infected roots.² First, plant genes could be turned on via a direct interaction between regulatory elements present in their promoters and nematode secretions. The putative expansin gene upa6 isolated from pepper (Capsicum annuum) is a homologue of LeEXPA5 and it is activated by bacterial type III effector AvrBs3.²⁷ Similarly, cyst nematodes might also posses a protein that is responsible for direct activation of expansin promoters. Second, the genes expression in infected roots could be triggered indirectly by plant hormones. Auxins, for example, are involved in syncytium induction and development and they seem to play a role in a preconditioning of cells prior to their fusion with the expanding syncytium.^28,29 Goellner and collaborators¹⁰ have already hypothesized the auxin-mediated activation of plant endo-β-1,4-glucanases upon syncytium development. Another tomato expansin gene upregulated in nematode infected roots, LeEXPA2,¹⁵ is expressed in auxin-treated hypocotyls,³⁰ but unfortunately nothing is known about hormonal regulation of expression of LeEXPA5 gene. However, it cannot be excluded that also LeEXPA5 gene is activated in nematode infected roots by elevated auxin levels.

Due to the fact that syncytia are the only source of nutrients for cyst nematodes, the generation of transgenic plants with silenced genes that are crucial for syncytium development is one of the possible approaches to engineer nematode resistant plants.³¹ In this context LeEXPA5 gene is of special interest, because it is specifically induced already in ISC, just at the onset of syncytium development and its transcript and protein distribution patterns suggest that it is involved in the process of syncytium hypertrophy.¹⁵ Constructing antisense lines or T-DNA mutants with silenced single members of multigene families often results in lack of clearly changed phenotype because of occurrence of functional redundancy.^32,33 Recent studies with tomato anti-sense lines harbouring construct silencing tomato endo-β-1,4-glucanases, which were specifically induced during cyst nematode infection, provided very promising results.²¹ Plant lines carrying anti-cel7 or anti-cel9C1 constructs did not exhibit abnormal phenotypes, but the nematode development on their roots was hampered. It suggests that while plants can overcome the knock down of some genes from multigene families by replacing silenced member by the others, the nematode induced cells seem to be less flexible. It is reasonable to avoid RNAi expression in other plant tissues and to limit it to nematode feeding site only by using syncytium specific promoters, e.g., promoter of AtSUC2,³⁴ which on the other hand is also active in phloem companion cells. Generated tomato lines transformed with anti-LeEXPA5 constructs show decreased susceptibility in nematode infection tests that reflects in lower number of developing adult females in comparison to susceptible tomato plants. Comparative microscopic analyses of syncytia induced in susceptible tomato and in transgenic tomato lines indicate high number of syncytia containing degenerated protoplasts already during early stages of syncytium development. Anatomical comparisons of syncytia induced in both genotypes show that 10 day old syncytia containing non-degenerated protoplasts are composed of fewer and less hypertrophied elements (Fig. 1B and C). In addition, the number of cell wall openings is lower and their sizes are smaller in syncytia induced in transgenic roots.

Addendum to: Fudali S, Janakowski S, Sobczak M, Griesser M, Golinowski W, Grundler FMW. Two tomato α-expansins show distinct spatial and temporal expression patterns during development of nematode induced syncytia. Physiol Plant. 2008;132:370–383. doi: 10.1111/j.1399-3054.2007.01017.x.