Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.

细胞间隙通常是病原菌最先侵染的位点。我们在感染烟草花叶病毒（TMV）并形成坏死斑的普通烟草（Nicotiana tabacum L.）叶片的细胞间隙中，检测到酸性病程相关（pathogenesis-related, PR）蛋白诱导剂发生积累。该诱导活性可从具有碱性特性的低分子量凝胶过滤组分中回收，且该组分中可洗脱得到纯品精胺（Spm）。我们对产生坏死斑的烟草叶片细胞间隙中的多胺进行了定量分析，发现游离精胺的含量较健康叶片高出20倍。在测试的多种多胺中，外源施加的精胺能够诱导酸性PR-1基因的表达。免疫印迹分析结果显示，精胺处理不仅提升了酸性PR-1蛋白的积累水平，还同时增加了酸性PR-2、PR-3及PR-5蛋白的积累量。对健康烟草叶片施加水杨酸（SA）后，内源精胺的含量未出现显著升高；而精胺处理也未使内源水杨酸的水平发生明显变化，这表明精胺诱导酸性PR蛋白的通路不依赖水杨酸信号途径。精胺处理还可缩小TMV诱导产生的局部病斑的尺寸。上述结果表明，病斑形成后，精胺会在细胞外积累，并通过不依赖水杨酸的信号通路同时诱导酸性PR蛋白的表达以及对TMV的抗性。