Cytology of infection of 35S-bean chitinase transgenic canola plants by Rhizoctonia solani: Cytochemical aspects of chitin breakdown in vivo

Abstract

Transgenic canola plants containing high, constitutive levels of bean endochitinase have been shown to be more resistant to infection by the soil-borne pathogen, Rhizoctonia solani, than are wild-type plants that lack the chimeric chitinase gene. To determine whether the resistance of the 35S-chitinase plants to Rhizoctonia infection results from an antimicrobial activity of the bean chitinase in planta, an ultrastructural and cytochemical study was performed on infected control and transgenic canola plants. Analysis of root tissues of infected wild-type canola plants revealed that R. solani was capable of extensive tissue colonization including the xylem vessels. Pathogen ingress towards the vascular system was associated with marked host cell wall alterations such as disruption of middle lamella matrices that occurred in advance of fungal penetration. Fungal hyphae colonizing these tissues appeared metabolically active as judged by their typical morphological features and their extensive multiplication. In infected transgenic plants, however, the pattern of fungal colonization was different to that observed in wild-type plants. Penetration of the host cuticle and epidermis was frequently observed, but fungal colonization was usually restricted to the cortex although, in a few cases, some fungal cells could be seen in xylem vessels. In all samples examined, severe hyphal alterations ranging from increased vacuolization to cell lysis were seen. Hyphae occasionally seen in xylem vessels were markedly damaged and often reduced to convoluted wall fragments. Cytochemical labeling of chitin using the WGA/ovomucoid-gold complex showed that hyphal alterations correlated with extensive chitin degradation. Thus, reduction in fungal biomass, increase in hyphal alterations leading to fungal lysis and chitin breakdown appear to be typical features observed in transgenic canola plants. Because these features were not seen in infected wild-type plants, it is likely that constitutive expression of the bean endochitinase gene is, at least in part, responsible for the enhanced protection against fungal attack observed in these plants. It is not known, however, if other components of the host defense response contribute to the resistance phenotype.