Dynamic hyphal specialization remodeling enables Botrytis cinerea to overcome diverse host defenses during infection

Abstract

Botrytis cinerea is a necrotrophic pathogen responsible for gray mold disease broadly devastating fruits, vegetables, and ornamental crops both in field production and postharvest supply chains. It can form multiple specialized hyphal morphotypes, but their cellular characteristics and functional significance remain inconsistently recognized. This study aimed to elucidate the cytological characteristics, developmental dynamics, and functional roles of specialized hyphal structures in enabling B. cinerea to adapt to hosts exhibiting varying resistance levels. Utilizing live-cell imaging and electron microscopy, it's demonstrated that B. cinerea forms non-canonical appressoria (termed appressoria-like structures, AP-Ls) after conidial germination. Unlike typical appressoria, AP-Ls lack melanin deposition and are independent of conidial autophagy. Although AP-Ls may normally mediate initial attempts to invade the host, plant cell death could be rarely observed around the fungal AP-L structure. As infection progresses, the hyphal tips could further differentiate into multicellular infection cushions (ICs), which contain intensified nuclei and organelles. Via visualizing the GFP-tagged nuclei and histochemical assays, it's shown that ICs exhibit enhanced tolerance to host-derived antimicrobial compounds than AP-Ls and non-differentiated hyphal cells. Additionally, IC development positively correlates with host resistance levels, being preferentially formed on recalcitrant hosts and triggering localized plant cell death nearby the IC formation sites. Notably, the ATP-binding cassette transporter BcAtrB, critical for phytoalexin efflux and fungicide resistance, appears to be upregulated within ICs during plant infection but not in the camalexin-deficient mutant pen3pdr12 as evidenced by GFP reporter assays, probably linking IC function to counteraction of host-derived defense chemicals. Our findings propose that B. cinerea AP-Ls are normally involved in initial host colonization, while multicellular ICs serve as adaptive hubs enabling the fungus to counteract robust defense chemicals derived from the hosts, thus the dynamic hyphal specialization remodeling could well underpin the success of this generalist pathogen in overcoming diverse plant defenses.