The apple U-box E3 ubiquitin ligase MpPUB23 enhances the resistance of apples to the fungal pathogen Penicillium expansum
苹果U框E3泛素连接MpPUB23增强抵抗的苹果到真菌病原菌的青霉expansum

The MpPUB23 protein is an unstable, hydrophilic, non-transmembrane, and it does not contain a signal peptide. The MpPUB23 protein features a U-box structural domain. The expression of MpPUB23 was induced by infestation through P. expansum. The subcellular localization of transiently transformed tobacco cells through Agrobacterium-mediated methods proved that the MpPUB23 protein was localized in the nucleus of the cell. Apple fruits overexpressing MpPUB23 showed an increased resistance to P. expansum by the transient transformation of apple fruits mediated by Agrobacterium, which revealed that MpPUB23 was a positive regulator of apple defense against P. expansum. The activity of defense-related enzymes and expression of related defense genes of apple fruits overexpressing MpPUB23 were measured, and it was found that MpPUB23 enhanced the resistance of apples to P. expansum by increasing the activity of defense-related enzymes and regulating the expression of defense genes. Changes in the expression levels of related genes after overexpression of MpPUB23 in apples were investigated by transcriptome sequencing. The results showed that the differentially up-regulated genes were mainly concentrated in the oxidoreductase activity and the biological pathway of oxidoreductase process. Furthermore, the differentially down-regulated genes were mainly enriched in the binding pathway. After overexpression of MpPUB23 gene, the expression of CHI2 and transcription factors WRKY22 and WRKY29 were up-regulated, and the expression levels of Ca²⁺ signal transduction related genes were changed, which induced the up-regulation of defense related genes and improved the defense ability of apple.
该MpPUB23蛋白质是不稳定的，亲，非跨膜，它不包含一个信号肽。 该MpPUB23蛋白质设有一个U框结构领域。 表达的MpPUB23诱发侵扰通过P.expansum. 亚细胞的本地化的瞬时变烟草的细胞通过杆菌介导的方法证明，MpPUB23蛋白质是本地化的核心单元。 苹果果表达MpPUB23表现出更大的阻力，P.expansum由瞬时变换的苹果通过杆菌，这表明，MpPUB23是一个积极的调节器的苹果的防御P.expansum. 该活动的与国防有关的酶和表达相关国防的基因的苹果表达MpPUB23进行测量，它是发现，MpPUB23增强抵抗的苹果P.expansum通过增加活动的与国防有关的酶和规范的表达，国防的基因。 变化的表达水平有关的基因后表达的MpPUB23在苹果进行了调查，通过转录组测序。 结果表明，有差别地上调基因主要集中在的氧化还原酶的活性和生物途径的氧化还原酶的过程。 此外，差异下调基因的主要富集在结合的途径。 之后表达的MpPUB23的基因的表达的CHI2和转录因素WRKY22和WRKY29上调，并表达水平的Ca ²⁺ 信号转导相关的基因被改变，这引起了监管的国防相关的基因并改进防御能力的苹果。

图 1 MpPUB23蛋白亲水性/疏水性（A）、跨膜结构域（B）和信号肽（C）预测

Fig. 1 Prediction of hydrophilicity/hydrophobicity (A), transmembrane domain (B) and signal peptide (C) of MpPUB23 protein
图。 1预测的亲/疏水性(A)、跨领域(B)和信肽(C)MpPUB23蛋白质

（A）结构域；（B）二级结构预测；（C）三级结构预测。

Fig. 2 Prediction of the structure of MpPUB23 protein
图。 2预测的结构MpPUB23蛋白质

(A) DNA binding region; (B) Secondary structure prediction; (C) Tertiary structure prediction.
(A)DNA结合地区；(B)二级结构的预测；(C)三级结构的预测。

图 3 P. expansum K1侵染苹果果实不同时间点MpPUB23的相对表达量

Fig. 3 Relative expression of MpPUB23 of apple fruits infected with P. expansum K1 for different hours
图。 3的相对表达MpPUB23的苹果的感染P.expansum K1对于不同的时间

图 4 MpPUB23蛋白在本氏烟草细胞中的亚细胞定位

Fig. 4 Subcellular localization of MpPUB23 protein in Nicotiana benthamiana cells
图。 4亚细胞化MpPUB23蛋白烟草本生烟细胞

注：转染融合35S∷eGFP质粒和转染35S∷MpPUB23-eGFP质粒的本氏烟草细胞的eGFP荧光，从左到右依次为：明场、核染色、绿色荧光和合并后的图像。

图 5 MpPUB23在瞬时转化的苹果果实中的相对表达量

图 6 pCambic1300和MpPUB23-OE苹果果实接种P. expansum K1 4、5、6和7天后的腐烂情况（A）、腐烂直径（B）和腐烂率（C）

Fig. 6 Decay (A), lesion diameter (B) and decay rate (C) of pCambic1300 and MpPUB23-OE apple fruits infected with P. expansum K1 for 4, 5, 6 and 7 days
图。 6衰变(A)、病变径(B)和衰减率(C)pCambic1300和MpPUB23-OE苹果水果感染P.expansum K1 4、5、6和7天

图 7 接种P. expansum K1后过表达MpPUB23对苹果SOD（A）、PPO（B）、PAL（C）活性的影响

Fig. 7 Effect of overexpression of MpPUB23 on SOD (A), PPO (B) and PAL (C) activity of apples inoculated with P. expansum K1
图。 7效果表达的MpPUB23在SOD(A)、PPO(B)以及PAL(C)活动的苹果接种P.expansum K1

注：P1300为对照，即用空载农杆菌转化的苹果，MpPUB23-OE为MpPUB23过表达瞬时转化的的苹果。

图 8 接种P. expansum K1后过表达MpPUB23对苹果相关抗病基因表达量的影响（3 d）

Fig. 8 Effect of overexpression of MpPUB23 on expression of related resistance genes of apples inoculated with P. expansum K1 (3 d)
图。 8影响表达的MpPUB23上表达的相关性基因的苹果接种P.expansum K1(3d)

图 9 差异表达基因数目统计

Fig. 9 Statistics of the number of differentially expressed genes
图。 9统计数字的差异表达的基因

注：A：数目统计；B：火山图（黄色标记了下调表达基因的分布情况，红色标记了上调表达基因的分布情况，而蓝色则代表了无显著差异基因的分布情况）。

图 10 苹果DEGs的GO二级分类通路富集分析

Fig. 10 Secondary classification pathway enrichment analysis of GO in apple DEGs
图。 10个次级分类的途径富集分析进去的苹果差异表达基因富集

图 11 苹果DEGs富集到的前10 GO三级分类通路

A：上调表达基因的GO功能富集分析；B：下调表达基因的GO功能富集分析

Fig. 11 The top 10 enriched GO tertiary classification pathways of apple DEGs
图。 11顶10富去三级分类途径的苹果差异表达基因富集

A:GO functional enrichment analysis of up-regulated; B: GO functional enrichment analysis of down-regulated genes.
答：进入功能的富集分析的上调；B：去功能的富集分析的下调的基因。

图 12 苹果DEGs的KEGG二级分类通路富集分析

Fig. 12 Secondary classification pathway enrichment analysis of KEGG in apple DEGs
图。 12辅助分类的途径富集分析所未有的大数据中的差异表达基因富集的苹果

图 13 苹果DEGs的KEGG富集分析

Fig. 13 KEGG enrichment analysis of DEGs in apples
图。 13所未有的大数据丰富的分析的差异表达基因富集中的苹果

图 14 MAPK信号转导、苯丙烷生物合成和植物-病原菌互作通路

Fig. 14 Pathway of MAPK signaling transduction of plant, phenylpropanoid biosynthesis and plant-pathogen interaction
图。 14途径的酶信号转导植物、苯丙生物合成和植物的病原体相互作用