Genome-wide identification and expression analysis of the pear autophagy-related gene PbrATG8 and functional verification of PbrATG8c in Pyrus bretschneideri Rehd.

MAIN CONCLUSION: Genome-wide identification, tissue-specific and stress expression analyses and functional characterization of PbrATG8s genes were conducted and the role of PbrATG8c in Botryosphaeria dothidea resistance was further investigated. Autophagy plays an important role in plant growth, development and stress tolerance. ATG8 has been reported to be an autophagy marker in many species. However, there is little information regarding ATG8 family members in pear (Pyrus bretschneideri Rehd). We performed a genome-wide analysis and identified nine PbrATG8 gene family members in pear. Phylogenetic analysis showed that PbrATG8 genes clustered into four major groups (Groups I-IV). Eight PbrATG8 genes were successfully mapped to 6 of the 17 chromosomes of the pear genome. The synteny results showed that two pairs are collinear. Gene expression data showed that all genes were differentially expressed in a range of pear tissues. Transcript analysis of PbrATG8 genes under dehydration, salt and pathogen infection stresses revealed that PbrATG8c responded to all test stresses. The PbrATG8c protein was localized in the nucleus and membrane. The silencing of PbrATG8c decreased the resistance to Botryosphaeria dothidea in pear. This study provides insights and rich resources for subsequent investigations of autophagy in pear.

Transcriptome Analysis of Pear Leaves in Response to Calcium Treatment During Botryosphaeria dothidea Infection.

Pear (Pyrus bretschneideri), one of the most widely planted fruit trees in the world, is infected by pear ring rot disease, which is triggered by Botryosphaeria dothidea. Previous research has shown that exogenous calcium enhanced pear resistance to B. dothidea. To explore the molecular mechanism of calcium in pear pathogen resistance, we searched the differentially expressed genes (DEGs) between calcium and H2O treatment with B. dothidea inoculation in pear by using RNA-seq data. On the basis of the standard of a proportion of calcium/H2O fold change >2, and the false discovery rate (FDR) <0.05, 2,812 and 572 genes with significant differential expression were identified between the H2O and calcium treatments under B. dothidea inoculation at 2 days postinoculation (dpi) (D2) and 8 dpi (D8), respectively, indicating that significantly more genes in D2 responded to calcium treatment. Results of the gene annotation showed that DEGs were focused on plant-pathogen interactions, hormone signal transduction, and phenylpropanoid biosynthesis in D2. Moreover, transient silencing of PbrCML30 (pear calmodulin-like proteins 30), which had significantly higher expression in response to calcium than H2O treatments, conferred compromised resistance to B. dothidea. Exogenous calcium treatment slightly alleviated the symptoms of TRV2-PbrCML30 leaves compared with TRV2 leaves under inoculation, supporting its key role in pear resistance to B. dothidea. Overall, the information obtained in this study provides a possible mechanism of calcium in regulating pear resistance to B. dothidea.

Exogenous Calcium Improved Resistance to Botryosphaeria dothidea by Increasing Autophagy Activity and Salicylic Acid Level in Pear.

Pear ring rot, caused by Botryosphaeria dothidea, is one of the most serious diseases in pear. Calcium (Ca2+) was reported to play a key role in the plant defense response. Here, we found that exogenous calcium could enhance resistance to B. dothidea in pear leaves. Less H2O2 and O2- but more activated reactive oxygen species scavenge enzymes accumulated in calcium-treated leaves than in H2O-treated leaves. Moreover, the increased level of more ascorbic acid-glutathione was maintained by Ca2+ treatment under pathogen infection. The expression of core autophagy-related genes and autophagosome formations were enhanced in Ca2+-treated leaves. Silencing of PbrATG5 in Pyrus betulaefolia conferred sensitivity to inoculation, which was only slightly recovered by Ca2+ treatment. Moreover, the salicylic acid (SA) level and SA-related gene expression were induced more strongly by B. dothidea in Ca2+-treated leaves than in H2O-treated leaves. Taken together, these results demonstrated that exogenous Ca2+ enhanced resistance to B. dothidea by increasing autophagic activity and SA accumulation. Our findings reveal a new mechanism of Ca2+ in increasing the tolerance of pear to B. dothidea infection.

Revealing the early response of pear (Pyrus bretschneideri Rehd) leaves during Botryosphaeria dothideainfection by transcriptome analysis.

Ring rot disease, which is caused by Botryosphaeria dothidea (B. dothidea), is one of the most serious diseases affecting the pear industry. Currently, knowledge of the mechanism about pear-pathogen interactions is unclear. To explore the early response of pear leaves to B. dothidea infection, we compared the early transcriptome of pear leaves infected with B. dothidea. The results revealed 3248 differentially expressed genes (DEGs) and 4862 DEGs at D2 and D4, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of DEGs showed that these genes were predominately involved in plant-pathogen interactions, hormone signal transduction and other biosynthesis-related metabolic processes, including glucosinolate accumulation and flavonoid pathway enhancement. However, many hormone- and disease resistance-related genes and transcription factors (TFs) were differentially expressed during B. dothidea infection. These results were consistent with the changes in the physiological characteristics of B. dothidea. In addition, the expression of PbrPUB29, an E3 ubiquitin ligase with a U-box domain, was significantly higher than it was at 0 dpi. PbrPUB29 silencing enhanced the sensitivity of pear leaves to B. dothidea, reflected by more severe symptoms and higher reactive oxygen species (ROS) content in the defective pear seedlings after inoculation, revealing that PbrPUB29 has a significant role in pear disease resistance. In brief, we explored the interaction between pear leaves and B. dothidea at the transcriptome level, implied the early response of pear leaves to pathogens, and identified a hub gene in a B. dothidea-infected pear. These results provide a basis and new strategy for exploring the molecular mechanisms underlying pear-pathogen interactions and disease resistance breeding.