The cell walls of syncytia formed by Heterodera schachtii in Arabidopsis thaliana are abundant in methyl-esterified pectin

Abstract

Plant-parasitic cyst nematodes form a specialized feeding site, termed a syncytium, in the roots of host plants. Monoclonal antibodies to defined glycans, in addition to a cellulose-binding module, were used to characterize the cell walls of a functioning syncytia in situ. Cell walls of syncytia were found to contain cellulose, xyloglucan and mannan. Analysis of the pectin network revealed syncytial cell walls are abundant in homogalacturonan, which was heavily methyl-esterified. Arabinan was also detected and the results suggest the cell walls of syncytia are highly flexible.

Keywords: :

• Arabidopsis thaliana
• Heterodera schachtii
• fluorescent imaging
• syncytia
• cell walls

Plant-parasitic cyst nematodes, such as Heterodera schachtii, form a specialized feeding site in the roots of host plants through which the nematode obtains plant-derived nutrients. The feeding site, termed a syncytium, is formed from a single vascular cylinder root cell, generally a procambial or pericycle cell (for review see ref. 1). The development of syncytia is associated with proliferation of procambial cells that are subsequently incorporated into the growing feeding site through extensive cell wall remodelling and cell wall degradation processes.¹ Many previous studies have focused on elucidating the cell wall remodelling processes that occur during syncytial development.^2-4 However, little was known about the structural architecture of syncytial cell walls. We recently used monoclonal antibodies to defined glycans and a cellulose-binding module to characterize the cell walls of syncytia formed by H. schachtii developing in A. thaliana.⁵ Transverse sections were taken through syncytia at 14 d post inoculation (dpi) and the cell wall architecture of syncytia and surrounding stele cells was characterized in situ.

Fluorescent imaging was used to analyze cellulose, hemicelluloses (xyloglucan and mannan) and pectin (homogalacturonan and rhamnogalacturonan-I), which constitute the carbohydrate component of plant cell walls. Additionally, the cell wall structural proteins arabinogalactan proteins (AGPs) and extensins were investigated. The immunolabelling of cellulose, xyloglucan and mannan (using CBM3a; LM15 and LM21antibodies respectively) were widely similar in sections taken through nematode infected root sections in comparison to uninfected roots. Cellulose was detected in cell walls of the syncytia (Fig. 1a) along with xyloglucan and mannan. It has been proposed that the hemicelluloses form cross-linkages with the cellulose microfibrils and provide the load-bearing structure in the syncytial cell wall.⁶

Figure 1. Fluorescent imaging of syncytial cell wall components. (A) Cellulose-binding module 3a (CBM3a) was used to visualize crystalline cellulose in the cell walls of syncytia formed by H. schachtii in A. thaliana at 14 dpi. (B) Transverse sections through nematode infected roots were pre-treated with 0.1 M sodium carbonate to remove methyl ester groups from pectin HG chains. Following pre-treatment the sections were treated with the LM19 antibody, which bound to the cell walls across the stele. (C) Immunolabelling with the LM20 antibody, which binds to methyl-esterified pectin HG resulted in strong fluorescence in all cell walls. Scale bars, 50 µm.

A range of monoclonal antibodies were used to characterize the pectin network of syncytial cell walls (Table 1). Pectin refers to a group of complex polysaccharides which includes homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). The antibodies LM19 and LM20 were used to localize pectin HG in nematode infected root sections and determine the methyl-esterification status of the polysaccharide. The LM19 antibody preferentially binds to de-esterified pectin HG⁷ and in nematode infected root sections the epitope was detected in pericycle cells only. However, immunolabelling with the LM19 antibody following the pre-treatment of root sections with 0.1 M sodium carbonate, which removes methyl esters from HG, resulted in strong binding of the antibody in all cells within the stele of nematode infected root sections (with the exception of xylem vessels; Figure 1b). This was confirmed with immunolabelling of the LM20 antibody, which binds to methyl-esterified pectin HG.⁷ The LM20 antibody bound extremely strongly across the stele in nematode infected root sections (Fig. 1c). In uninfected root sections the LM20 epitope was detected at a low level. The LM19 and LM20 antibody immunolabelling collectively demonstrate that syncytial cell walls are abundant in pectin HG, which is heavily methyl-esterified. The pectin network is a major component of plant cell walls and the methyl-esterification status of pectin HG influences the mechanical properties and porosity of cell walls.⁸ There is a body of evidence that suggests homogalacturonan is synthesized in the Golgi apparatus where 70–80% of the galacturonic acid residues are methyl-esterified prior to secretion into the primary cell wall (for review see⁹). Methyl-ester groups are subsequently removed from regions of HG in the cell wall by the enzymatic action of pectin methylesterases.¹⁰ De-esterified regions of HG can form cross-linkages with calcium ions, which can reduce porosity and strengthen the cell wall.^11,12 The heavily methyl-esterified status of the pectin HG in the cell walls of syncytia is predicted to result in a highly flexible wall. Flexibility is an important property of syncytial cell walls as the cell must withstand high turgor pressures, which arise from solutes accumulating in the syncytia.¹³ Additionally, the feeding site must maintain structural integrity following the nematode ingesting the cytoplasmic contents of the syncytium.

Table 1. Overview of monoclonal antibodies used to characterize the structural architecture of syncytial cell walls together with details of antibody epitope detection.

Primary antibody	Cell wall component	Detected in cell walls of syncytia?
LM11	Xylan	X
LM15	Xyloglucan	√
LM21	Mannan	√
LM19	De-esterified pectin HG	√
LM20	Methyl-esterified pectin HG	X
LM5	Galactan	X
LM6	Arabinan	√
LM16	Processed arabinan	√
LM2	AGPs	X
JIM13	AGPs	√
JIM19	Extensin	X

While the highly methyl-esterified status of pectin HG provides an obvious function in terms of cell wall flexibility, it does raise questions with regards to cell wall degradation processes. For cell wall degradation to occur, the disassembly of all cell wall polymers is required. Pectin chains are hydrolysed by pectate lyases and polygalacturonase enzymes however, these enzymes are unable to act upon pectin HG which is methyl esterified (for review see¹⁴). During fruit ripening, a process that requires extensive cell wall degradation, the de-esterification of methyl-esterified HG is an important step in the degradation process.^15-17 Therefore, at 14 dpi the syncytium is a cell in which extensive cell wall degradation has occurred but pectin, one of the major polysaccharide components of the cell wall, is in a condition that is protected from degradation. We characterized syncytia the feeding size had reached the maximum size, as a result we revealed the cell wall structural architectural requirements for syncytial function as opposed to formation. We will now go on to determine the methyl-esterification status of pectin HG during the early stages of syncytial development, when the extensive cell wall remodelling and degradation processes are occurring. This will hopefully provide an insight into how the dynamic cell wall adapts to these conflicting requirements.

Abbreviations:
dpi	=	days post inoculation
HG	=	homogalacturonan

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.