Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation.

Francis Onyilo,Li-Hung Chen,Wilberforce Tushemereirwe,Ioannis Stergiopoulos,Jaindra N Tripathi,Jerome Kubiriba,Geoffrey Tusiime,Leena Tripathi,Bryce Falk,Charles Changa

doi:10.3389/fmicb.2017.00830

Abstract

Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijiensis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1-silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

Highlights

Banana and plantain (Musa sp.) is the eighth most economically staple food crop (Tripathi et al, 2014)
The RNA interference (RNAi) construct was validated by confirming the presence of PfHog1 gene through end point PCR using the primers designed from the promoters region of pKOIISD1 plasmid construct
The sense strand of PfHog1 gene was driven by PtrpC promoter and its anti-sense strand was regulated by Pgpd promoter (Figure 1)

Summary

Introduction

Banana and plantain (Musa sp.) is the eighth most economically staple food crop (Tripathi et al, 2014) Worldwide it is cultivated across tropical and subtropical countries on more than 10 million hectares and total production estimated at 144 million tons (FAOSTAT, 2013). It is one of the fundamental energy sources for millions of people in developing countries and the most significant of all fruits, with world trade total of $2.5 billion annually. In Africa, banana and plantain are mainly used as a staple food, providing more than 25% of the carbohydrate requirements for over 70 million people (IITA, 1998) It is a critical and often the only source of income for both small and large-scale farmers, being an important contributor to local economies. Banana is the most important staple food crop in Uganda, its production is declining since 2010 (FAOSTAT, 2013), probably due to pests and diseases

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