Infection by cyst nematodes induces rapid remodelling of developing xylem vessels in wheat roots

Abstract

Cyst nematodes induce host-plant root cells to form syncytia from which the nematodes feed. Comprehensive histological investigation of these feeding sites is complicated by their variable shape and their positions deep within root tissue. Using tissue clearing and confocal microscopy, we examined thick (up to 150 μm) sections of wheat roots infected by cereal cyst nematodes (Heterodera avenae). This approach provided clear views of feeding sites and surrounding tissues, with resolution sufficient to reveal spatial relationships among nematodes, syncytia and host vascular tissues at the cellular level. Regions of metaxylem vessels near syncytia were found to have deviated from classical developmental patterns. Xylem vessel elements in these regions had failed to elongate but had undergone radial expansion, becoming short and plump rather than long and cylindrical. Further investigation revealed that vessel elements cease to elongate shortly after infection and that they later experience delays in secondary thickening (lignification) of their outer cell walls. Some of these elements were eventually incorporated into syncytial feeding sites. By interfering with a developmental program that normally leads to programmed cell death, H. avenae may permit xylem vessel elements to remain alive for later exploitation by the parasite.

Figure 1

Method to image and model thick sections of control and infected wheat root tissue. (a) Root tissue was embedded and cut into 150 µm longitudinal sections. (b) Sections containing the central part of the vascular bundle were chosen. (c) Sections were stained with calcofluor white and propidium iodide and then imaged using a confocal microscope and processed with Nikon NIS-Elements to produce a maximum projected image. (d) Using IMARIS software, the central metaxylem vessel was manually selected and highlighted in order to render and (e) rotate a three-dimensional surface model of the central metaxylem. This same process was used to analyse wheat seminal root tissue at 15 d after inoculation with cereal cyst nematodes. (f) Maximum projected confocal images stained and showing facing surfaces of two consecutive sections containing a nematode (N), a central metaxylem (cMX) vessel and a feeding site (FS). (g) Features manually selected and coloured: the nematode in red, the feeding site in blue and the central metaxylem in green (three vessel elements that have been ‘invaded’ by the feeding site) and yellow. (h) Three-dimensional models of the selected features. A detailed rotation of the left-hand model is shown in Supplementary Video S1. (VC) vascular cylinder (cMX) central metaxylem, (LRI) lateral root initial, (COR) cortex. Scale bar (a–d) 200 µm (f–h) 100 µm.

Figure 2

Central metaxylem development in wheat seminal roots with and without infection by cereal cyst nematodes. (a–d) The upper panel shows longitudinal sections from control (mock-inoculated) roots and the lower panel shows longitudinal sections of inoculated roots at (a) 2 d after inoculation (DAI), (b) 7 DAI, (c) 15 DAI, and (d) 21 DAI. Left-hand panels show maximum projection confocal images (processed with Nikon NIS-Elements) of tissue stained with calcofluor white (blue) and propidium iodide (red). Right-hand panels show the corresponding three-dimensional models (produced using IMARIS) of central metaxylem (cMX) vessels, coloured yellow and tilted to show vessel end walls. (e,f) Development of cereal cyst nematode feeding sites and xylem connectivity in wheat seminal roots. (e) A feeding site and a cMX vessel at 7 d after inoculation (DAI), (f) a feeding site and two peripheral metaxylem (pMX) vessels at 15 DAI and (g) a feeding site and a pMX vessel at 21 DAI. Each upper panel shows maximum projection confocal images (processed with Nikon NIS-Elements) of the facing surfaces of two consecutive sections of tissue stained with calcofluor white (blue) and propidium iodide (red). Each lower panel shows isolated three-dimensional models (produced using IMARIS) of feeding sites in aqua and xylem vessels in green (vessel elements that have been ‘invaded’ by the feeding site) and yellow. Rotation of the models is shown in Supplementary Video S2. Scale bar 50 µm.

Figure 3

Transverse sections of wheat seminal roots treated with a lignin-specific stain. The upper panels show sections of control (mock-inoculated) roots. The lower panels) show sections of roots inoculated with cereal cyst nematodes. All sections were stained with basic fuchsin (red) and calcofluor white (blue), imaged using a confocal microscope and processed with Nikon NIS-Elements to produce a maximum projected images. (a) 2 d after inoculation (DAI), (b) 4 DAI, (c) 6 DAI (d) 8 DAI. (LRI) lateral root, (FS, circled in white) feeding site, (pMX) peripheral metaxylem, (cMX) central metaxylem. Scale bars: 100 µm.

Figure 4

Proliferation of lateral root formation on wheat seminal roots infected with cereal cyst nematodes. (a) A control (mock-inoculated) root, with five emerging lateral roots. (b) A root at 7 d after inoculation with cereal cyst nematodes, with three advanced lateral roots above the infection site and many lateral roots emerging at the infection site. Scale Bar: 500 µm.

Figure 5

Experimental setup and growth hydroponics system. (a) Seedlings germinated and inoculated on agar plates. (b) Seedling transferred to Eppendorf tube with tip removed. (c) Tube placed into a 50 mL tube with base removed. (d) Tubes arranged in an aerated hydroponic tank containing nutrient solution.