Phytoplasmas are insect-borne plant pathogenic bacteria that alter host morphology. TENGU, a small peptide of 38 residues, is a virulence factor secreted by phytoplasmas that induces dwarfism and witches' broom in the host plant. In this study, we demonstrate that plants process TENGU in order to generate small functional peptides. First, virus vector-mediated transient expression demonstrated that the amino-terminal 11 amino acids of TENGU are capable of causing symptom development in Nicotiana benthamiana plants. The deletion of the 11th residue significantly diminished the symptom-inducing activity of TENGU, suggesting that these 11 amino acids constitute a functional domain. Second, we found that TENGU undergoes proteolytic processing in vitro, generating peptides of 19 and 21 residues including the functional domain. Third, we observed similar processing of TENGU in planta, and an alanine substitution mutant of TENGU, for which processing was compromised, showed reduced symptom induction activity. All TENGU homologs from several phytoplasma strains possessed similar symptom induction activity and went through processing, which suggests that the processing of TENGU might be related to its function.

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We produced transgenic Nicotiana plumbaginifolia plants which contained Spo min (sporamin minimal promoter)-GUS fused chimeric gene constructs with 5 types of signal sequences, such as cytosol, apoplast, ER, vacuole and plastid, and analyzed the GUS expression patterns after sucrose treatment. Spo min induced extremely high GUS activities after 6% and 10% sucrose treatment, especially in leaves. The high GUS activities were observed in leaves of the Spo min -ER-GUS construct treated with 6% or 10% sucrose. These were over 200 times higher than those in leaves with the 35S promoter-ER-GUS construct. The 10% sucrose treatment significantly altered GUS activities in all Spo min and 35S promoter constructs compared with those in the 6% sucrose treatment; some increased and some decreased. GUS activities in 2 months old plants were almost the same as 8 months old plants, indicating that GUS expression driven by Spo min was stably maintained. Also, even when sucrose treatment was stopped, GUS gene expression by Spo min continued for 10 days.

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