In this study, we used Agrobacterium tumefaciens -mediated transient expression system in Nicotiana benthamiana and confocal microscopy to investigate the immunoregulation roles and subcellular localization of CcCAP1, a virulence-related effector identified in C.

CcCAP1 suppressed the programmed cell death triggered by Bcl-2-associated X protein (BAX) and the elicitin infestin1 (INF1) in transient expression assays with Nicotiana benthamiana.

The CAP superfamily domain was sufficient for its cell death-inhibiting activity and three of the four cysteines in the CAP superfamily domain were indispensable for its activity.

In addition, expression of green fluorescent protein-labeled CcCAP1 in N.

These results suggest that this virulence-related effector of C.

IMPORTANCE The data presented in this study provide a key resource for understanding the biology and molecular basis of necrotrophic pathogen responses to Nicotiana benthamiana resistance utilizing effector proteins, and CcCAP1 may be used in future studies to understand effector-triggered susceptibility processes in the Cytospora chrysosperma -poplar interaction system.

INTRODUCTION Cytospora chrysosperma, a pathogenic fungus that causes canker disease, attacks nearly 80 species of woody plants, including poplar ( Populus sp.), causing serious forestry and ecological damage each year, especially in China ( 1 4 ).

Until now, studies on this disease have been limited to epidemiology, histocytology, and taxonomy ( 5 7 ), with few molecular biology studies in progress ( 8 ).

However, understanding the molecular mechanisms of pathogenesis is important for the development of strategies for durable and efficient control of plant diseases ( 9, 10 ).

Plants possess a multifaceted innate immune system to guard themselves against phytopathogens ( 11 ).

Pathogen-associated molecular patterns (PAMPs) induce PAMP-triggered immunity (PTI), the first layer of plant immunity known as a type of basal defense ( 12 ).

PAMPs are evolutionarily conserved molecules, such as lipopolysaccharide, the translation elongation factor EF-Tu, and flagellin from bacteria ( 13, 14 ); -glucan, chitin, and ergosterol from fungi ( 15 17 ); and transglutaminase GP42, cellulose-binding elicitor lectin, and the elicitin infestin1 (INF1) from oomycetes ( 18 20 ).

PAMPs are often recognized by plants via pattern recognition receptors on the plasma membrane, including receptor-like kinases, receptor-like proteins, and receptor-like cytoplasmic kinases ( 21 23 ).

This basal defense response can restrict the proliferation of most pathogens via callose deposition in the cell walls, reactive oxygen species (ROS) accumulation, and transcriptional upregulation of immune-related genes ( 23 ).

Furthermore, phytopathogens evade or overcome PTI for further colonization in the host by delivering effectors into the plant cytoplasm or apoplastic space ( 24 29 ).

However, when effectors are recognized by corresponding resistance (R) proteins in the host plants, effector-triggered immunity is induced, which is a qualitatively swifter and more vigorous immune response than PTI and induces localized programmed cell death (PCD) in the host, also called the hypersensitive response (HR) ( 30 ).

Dedicated research on effector functions is important to understand the pathogenesis of phytopathogens and to contribute to breeding efforts for improved protection from disease ( 31 ).

The past few decades have seen great progress in our knowledge of the activity of effectors and targets in host plants, which revealed that effectors manipulate plant immunity in several ways ( 32 36 ).

For example, some effectors suppress the RNA silencing process in host plants.

For instance, Phytophthora suppressors of RNA silencing 1 and 2 (PSR1 and PSR2, respectively) from Phytophthora sojae inhibited the production of small RNA to hinder plant resistance ( 37 39 ).

Some effectors interfere with PTI.

Fungal effectors can be used according to their different pathogenic strategies for the identification of key components of plant innate immunity and for disease resistance breeding ( 36 ).

However, of the several hundred effectors potentially produced by each phytopathogen, only a few effectors have been functionally characterized.

The localization of effector proteins in host cells often provides important clues to their mode of action ( 36 ).

Dd Form 214-Automated Nov 88 Template