Biochemical aspects of resistance to rust by barley

Abstract

In this work the in vivo RNA synthesis in the inoculated leaves of barley Hordeum vulgare L. cvs. Kakapo, Zephyr and DSIR725-02, was investigated using the inhibitor Actinomycin-D (Act-D). The results indicated that the expression of susceptibility may not be dependent on the synthesis of RNA. In the incompatible interactions (KA/A11 and DS/PS) this inhibition, of in vivo RNA synthesis by Act-D, was accompanied by a rapid fungal growth, comparable to the compatible interactions, about 14-16h after inoculation.

The assessment of temperature effects on these interactions revealed that, for the Act-D treated leaves as compared to the controls, the higher temperature (26°C) seemed to favour more fungal growth than the lower temperature of 18°C.

A method, using high salt (400mM K⁺), high pH (9.00) and Triton X-100, was developed for the isolation of free polysomes from both the healthy and inoculated leaves of the three barley cultivars. It was found that between 20-30% more monosomes were present in the polysome extracts from the compatible interactions (KA/PS and ZE/PS) than in the incompatible interactions (DS/PS and KA/All). There were 57% more monosomes, for the compatible interaction KA/PS at 26°C, in the inoculated leaves than in the corresponding healthy leaves.

The large polysomes in the compatible interactions appeared to have been selectively hydrolysed during infection to produce small sized "mers". Such an action was characteristic of the compatible interactions and was often observed as early as 12h after inoculation. The increase in the large polysomes, for the incompatible interactions, occurred before 48h after inoculation.

In this work, too, the polysomal mRNA translation activity was investigated to establish the nature and extent of the effects, on the host gene expression, induced by the inoculation of the three barley cultivars with Puccinia hordei Otth isolate PS. Hence the polypeptides directed in vitro by the free polysomes were analysed by SDS-polyacrylamide slab gel electrophoresis and liquid scintillation counting.

The results have shown that rust infection appeared to suppress the synthesis of polypeptides, of the molecular weight sizes of 130Kd, 68Kd and 24Kd in the compatible interactions. Nevertheless increases were also noted in the 43Kd and 18Kd molecular weight ranges while free mRNA showed increases in the 43Kd MW region only. For the incompatible interactions the increases were in the regions of 130Kd and 28Kd. These changes, in the incompatible, were observed to occur by 12h after inoculation. Such swift action, of invoking rapid synthesis of polysomal mRNA in the resistant host plant, was interpreted to reflect a possible concerted defence mechanism 'harnessed ' to combat the fungal challenge.

In view of the possible origins of the in vitro synthesized polypeptides the organelle-specific inhibitor of protein synthesis (CAM) was used to ascertain indirectly the contribution of chloroplast genome expression to the overall polysomal mRNA changes in the barley-rust systems studied here. The results of in vitro translation, in the presence of CAM, showed that the polypeptides in the regions 80-92Kd, 53-65Kd and 24-28Kd may have originated from the chloroplasts. The rust infection suppressed, in the compatible interactions, the synthesis of the CAM-sensitive 28Kd products. While CAM-sensitivity, for the incompatible interactions, was evident in two more regions (150Kd and 80Kd) the rust infection was associated with increases in the two MW ranges of 80Kd and 28Kd. Furthermore the temperature effects, on the chloroplast-derived polypeptides, were less profound compared to those effects on the polypeptides derived from the cytoplasm. Therefore the indirect evidence, also presented in this work, supports the involvement of chloroplast genome in the expression of resistance to rusts by barley.