An extended N-terminus restrains the plant cell death-inducing ability of the catalytically competent ribonuclease domain in a pea powdery mildew RALPH effector

Sahu, D.; Ghosh, P.; Mukherjee, S.; Kumar, V.; Sharma, G.; Gupta, M.; Gupta, G.; Ray, P.; Kusum,; Sharma, J.; Jain, D.; Chandran, D.

doi:10.64898/2026.08.13.744637

bioRxiv · 10.64898/2026.08.13.744637

An extended N-terminus restrains the plant cell death-inducing ability of the catalytically competent ribonuclease domain in a pea powdery mildew RALPH effector

Sahu, D.·Ghosh, P.·Mukherjee, S.·Kumar, V.·Sharma, G.·Gupta, M.·Gupta, G.·Ray, P.·Kusum,·Sharma, J.·Jain, D.·Chandran, D.

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Abstract

RALPH (RNase-like proteins associated with haustoria) effectors, which are preferentially expressed in haustoria and structurally resemble fungal T1/F1 RNases, constitute one of the largest effector families in powdery mildew (PM) fungi, yet their functions in dicot-adapted PM species remain poorly understood. Unlike cereal PM RALPHs, which lack the catalytic residues required for RNase activity, some dicot PM RALPHs retain these residues. Here, we performed a comprehensive structural and expression-based characterization of the pea PM Erysiphe pisi RALPH (EpRALPH) repertoire and functionally characterized EpRALPH11, a RALPH effector with partial conservation of the catalytic residues of T1/F1 fungal RNases. Comparative analyses identified multi-RNase-domain RALPHs as a conserved feature of the Erysiphe lineage, while expression profiling showed that many EpRALPHs are preferentially expressed in haustoria during early host colonization. AlphaFold 3-based structural analyses revealed a conserved T1/F1 RNase-like fold despite substantial sequence and surface charge divergence, indicating functional diversification among EpRALPHs. EpRALPH11 enhanced susceptibility to E. pisi in Medicago truncatula, localized to the nucleolus, and induced nucleolar fragmentation when heterologously expressed in Nicotiana benthamiana leaves. Its RNase domain exhibited T1 RNase activity in vitro, supporting the retention of a catalytically competent RNase domain and, together with its nucleolar localization, suggesting that EpRALPH11 targets plant rRNA and disrupts nucleolar functions. The RNase domain induced cell death in N. benthamiana, whereas the full-length protein and catalytic mutants did not. Cell death induction required exclusive nucleolar localization of the RNase domain, and an extended N-terminal intrinsically disordered region suppressed this activity in the full-length protein. Together, our findings reveal a previously unrecognized mechanis regulating RNase activity in a dicot PM RALPH effector and provide new insights into the functional diversification of RALPHs and their adaptation to obligate biotrophy.

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Sahu, D., Ghosh, P., Mukherjee, S., Kumar, V., Sharma, G., Gupta, M., Gupta, G., Ray, P., Kusum,, Sharma, J., Jain, D., Chandran, D.. 2026-08-18. An extended N-terminus restrains the plant cell death-inducing ability of the catalytically competent ribonuclease domain in a pea powdery mildew RALPH effector. https://doi.org/10.64898/2026.08.13.744637

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