Destabilization of potato spindle tuber viroid by mutations in the left terminal loop

Area of Science:

• Plant Pathology
• Molecular Virology
• RNA Biology

Background:

• Potato spindle tuber viroid (PSTVd) is a significant plant pathogen.
• Understanding viroid structure-function relationships is crucial for disease control.
• Nonviable viroid mutants provide insights into essential structural requirements for infectivity.

Purpose of the Study:

• To investigate the biological properties of apparently nonviable PSTVd mutants.
• To determine the infectivity and recovery mechanisms of structurally compromised PSTVd variants.
• To elucidate the role of host factors and spontaneous mutations in viroid viability.

Main Methods:

• Infectivity studies using RNA inocula generated by ribozyme cleavage.
• Mechanical inoculation of tomato seedlings with PSTVd mutant RNA transcripts.
• Analysis of transgenic Nicotiana benthamiana expressing PSTVd mutants.
• In vitro structural analysis of PSTVd mutants.
• Reverse transcription-polymerase chain reaction (RT-PCR) for RNA detection.

Main Results:

• A PSTVd mutant (PSTVd-P) with nucleotide substitutions in the left terminal loop was noninfectious upon mechanical inoculation.
• Viable progeny with a spontaneous C-->G change at position 4 were recovered from transgenic plants expressing PSTVd-P.
• PSTVd-P exhibited weakened native structure in vitro and instability of the ribozyme-cleaved full-length molecule.
• No detectable circularized PSTVd-P was found in uninfected plants via RT-PCR.

Conclusions:

• The initial mutations in PSTVd-P significantly weakened its structure, leading to nonviability.
• Recovery of viable progeny suggests a complex interplay between host RNA polymerase II errors during transcription and strong selective pressure against structural alterations.
• Spontaneous mutations, like the C-->G change at position 4, combined with host transcription fidelity, are critical for restoring PSTVd viability.