Characterization and overexpression of RHO1 from Cryptococcus laurentii ZJU10 activates CWI signaling pathway on enhancing the inhibition of blue mold on pears

Highlights

• A RHO1 gene from C. laurentii ZJU10 was characterized for the first time.

• The RHO1 gene was cloned and overexpressed in S. cerevisiae.

• Rho1 highly reduced the blue mold on pears by activating CWI signaling pathway.

• Its mechanism of biocontrol was induction of host resistance.

Abstract

Results from this study explored the inhibitory effect of RHO1 gene (GenBank accession number KY859864) from the antagonistic yeast, Cryptococcus laurentii ZJU10, on the control of Penicillium expansum in pear fruit and its possible mechanism involved. The RHO1 gene was successfully cloned and overexpressed in Saccharomyces cerevisiae. Sequence analysis showed high similarity with Rho family proteins, implying a primary role of Rho1 in the cell wall integrity (CWI) signaling pathway. Gene expression of RHO1 and other five CWI-related genes (including Pkc1, Rlm1, Fks1, Fks2 and Chs3) were significant up-regulated in the treatment of SC/Rho1-induced strain (Saccharomyces cerevisiae transformed with RHO1 and induced by galactose). Meanwhile, SC/Rho1-induced treatment reduced about 61.5% of disease incidence and almost 5-times lower lesion diameter compared to the control. In addition, the growth of transformed strains was slightly lower in contrast to the wild Saccharomyces cerevisiae and the induction of fruit resistance was significantly enhanced, which was tightly linked with triggering stronger host defensive responses by priming activation. This is the first study that Rho1 has a potential function of suppressing fungal disease in harvested fruit by activating CWI signaling pathway and indicates an alternative strategy for postharvest disease management.

Introduction

Penicillium expansum is one of major pathogens of pear fruit worldwide (Sanchez et al., 2016), which causes considerable losses during transportation and postharvest storage period (Banani et al., 2014; Sharma et al., 2009). Besides the economic aspects, metabolite secreted by P. expansum leads the potential risk of human's health and environment, which has attracted more and more public attention. Moreover, persistent misuse of synthetic fungicides has caused increasing resistance of pathogens. Hence, the need to develop alternative control method has become increased (Nunes, 2012; Palou et al., 2016).

Cryptococcus laurentii has been shown great efficacy on controlling postharvest disease of various fruit, including anthracnose on mangos (Ocampo-Suarez et al., 2017), P. expansum on peaches (Zhang et al., 2017) and on apples (Jiangkuo et al., 2017). An extensiveness of modes of action have been proposed as mechanisms for biocontrol, such as competition for nutrients and space (Yu and Lee, 2015), secretion of lytic enzymes (Banani et al., 2015), induction of resistance (Lu et al., 2013), mycoparasitism and antibiosis (Pretorius et al., 2015; Zhong, 2012). Most of the works are focused on secretion of lytic enzymes and competition for nutrients and space, however, a clear understanding of molecular mechanism (including induction of resistance) is still difficult due to the lack of genetic background of antagonistic yeast and methods. Transcriptomic analysis by high-throughput sequencing technology makes it possible to study gene expression changes and provides an insight on the molecular mechanism, so as to accurately define and help improve the effectiveness of biological control.

The cell wall is the first barrier for fungal cells to connect with the outside world and has been demonstrated involved in reducing disease resistance in pear fruit (Sun et al., 2018). Rho1 is an essential protein that controls CWI signaling pathway which receives inputs from the cell surface and regulates activation of protein kinase C (Pkc1), organization of actin cytoskeleton, activity of β-1,3-glucan synthase (GS) and polarized secretion (Levin, 2005, Levin, 2011). Meanwhile, Delgado et al. (2016) demonstrated that Rho1 accumulation provides a complementary means to combat resistance to antifungal proteins. Nevertheless, few studies have been reported in the role of antagonistic yeast Rho1 in inhibition of fungal disease of harvested fruit.

The objective of this study was to assess RHO1 gene from C. laurentii ZJU10 overexpressed in S. cerevisiae on control of blue mold decay caused by P. expansum in pear fruit during the postharvest storage and the potential mechanism involved.