Investigations of barley powdery mildew effectors (CSEPs) in the non-host plant wheat and host plant barley

Abstract

Blumeria graminis f. sp. hordei is a biotrophic obligate fungus that can survive and cause powdery mildew disease exclusively on living barley plants. Over 500 Candidate Secreted Effector Proteins (CSEPs), which are considered to be secreted by the fungal feeding structure haustoria, have been identified. Some of the CSEPs, such as CSEP0064 and CSEP0264, were found to contribute to the pathogen virulence. Wheat is resistant to Blumeria graminis f. sp. hordei-This is a form of non-host resistance. Recently, a Pseudomonas fluorescens based effector-to host analyser strain (EtHAn) was developed as a modified T3SS, which can deliver foreign effectors into plants. In this study, I used the EtHAn strain to deliver CSEP0064 and CSEP0264 into wheat, and screened for effector-triggered responses in a mapping population of wheat (WAGTAIL). Although the bacterium itself induced pattern-triggered immunity (PTI) in wheat, three cultivars (Rialto, Abbot and Madrigal) were capable of consistently recognizing and differentiating introduced CSEP0064 or CSEP0264, leading to an enhancement of EtHAn-triggered PTI. Subsequently, I tested whether the recognition of EtHAn-delivered effectors in these three lines can affect the susceptibility of wheat to subsequent fungal infection. The results showed that the recognition of introduced CSEPs triggered systemic resistance in Rialto and Madrigal, whereas induced systemic susceptibility in Abbot, to the adapted pathogen Blumeria graminis f. sp. tritici. Delivered CSEPs did not affect systemic wheat immunity to sebsequent infection of the non-adapted pathogen Blumeria graminis f. sp. hordei. These results suggest that either wheat host resistance and non-host resistance are controlled by different systemic signalling pathways or basal plant immunity is sufficient to restrict non-host pathogen invasion and additional up-regulation of systemic immunity does not provide any additional advantage.

In parallel, the expression of CSEP0064 in host plant barley was also investigated. In this study, I observed that CSEP0064 is detectable by western blotting in heavily infected barley leaves and isolated haustoria. Unexpectedly, a protein with an apparent molecular mass around 200 kDa was recognized by the anti-CSEP0064 antibody on western blots. Here, I refer to this protein as ‘Big CSEP’. This ‘Big CSEP’ was detected in fungal structure-containing materials only. TFMS-mediated deglycosylation successfully removed this ‘Big CSEP’ signal, suggesting that the ‘Big CSEP’ may be a hyper/O-glycosylated CSEP0064, or a glycosylated complex containing CSEP0064. Large-scale shotgun proteomics have been performed on Blumeria graminis f. sp. hordei, leading to the identification of some haustoria-exclusive effectors including CSEP0064. Here, MRM-MS was used to detect and quantify CSEP0064 in different fractions of infected barley leaf. An enzymatic method was conducted to isolate haustoria from infected barley epidermis. GAPDH (Blumeria graminis f. sp. hordei) and GAPDH (H. vulgare) were used as reference for fungal and plant proteins. CSEP0064 was more abundant than GAPDH (Blumeria graminis f. sp. hordei) in isolated haustoria and the plant cytoplasm fractions. Moreover, CSEP0064 was four times more abundant than GAPDH (H. vulgare) in the plant cytoplasm fraction. Finally, the results demonstrate the secreation of CSEP0064 from haustoria and the uptake of CSEP0064 by the host cell.