Functional Analysis of the GH16 Domain-Containing XTH2 Homologs in Mediating Sunflower Response to Orobanche cumana...

Sunflower (Helianthus annuus) is highly susceptible to infection by the root parasitic plant Orobanche cumana during its growth. In establishing connections with the sunflower root system, O. cumana induces the hydrolysis and remodeling of the host cell wall. Xyloglucan endotransglucosylase/hydrolase (XTH), a member of glycoside hydrolase family 16 (GH16), is a key enzyme involved in the hydrolysis and synthesis of xyloglucan, playing a critical role in cell wall modification and reconstruction. However, the involvement of XTH families in the interaction between sunflower and O. cumana remains unclear. In this study, we showed that the expression level of HaXTH2 was upregulated in sunflowers following O. cumana infection. Overexpression of HaXTH2 loosens host primary cell walls and represses lignin-based defense responses at the early infection stage, thereby facilitating haustorial penetration across cortical tissues and xylem bridge formation to support normal parasitism of O. cumana. Functional analysis revealed that HaXTH2 and its homolog, HaXTH2-1, facilitate O. cumana infection, whereas OcXTH2, an O. cumana homolog, suppresses this process. Furthermore, HaXTH2, HaXTH2-1, and OcXTH2 are localized to the cell wall. Domain truncation analysis revealed that the GH16 domain alone from HaXTH2 and HaXTH2-1 enhance parasitic susceptibility, while the xyloglucan endotransglycosylase C-terminal domain (XET_C) domain does not. Mutation analysis identified Y20 in HaXTH2 and H96 in HaXTH2-1 as key amino acid sites regulating O. cumana parasitism. This study expands our understanding of the functions of XTHs in plant-plant interactions and provides a theoretical basis for further development of O. cumana-resistant sunflower cultivars.