Transcriptome analysis of Actinidia chinensis in response to Botryosphaeria dothidea infection

Ripe rot caused by Botryosphaeria dothidea causes extensive production losses in kiwifruit (Actinidia chinensis Planch.). Our previous study showed that kiwifruit variety “Jinyan” is resistant to B. dothidea while “Hongyang” is susceptible. For a comparative analysis of the response of these varieties to B. dothidea infection, we performed a transcriptome analysis by RNA sequencing. A total of 305.24 Gb of clean bases were generated from 36 libraries of which 175.76 Gb was from the resistant variety and 129.48 Gb from the susceptible variety. From the libraries generated, we identified 44,656 genes including 39,041 reference genes, 5,615 novel transcripts, and 13,898 differentially expressed genes (DEGs). Among these, 2,373 potentially defense-related genes linked to calcium signaling, mitogen-activated protein kinase (MAPK), cell wall modification, phytoalexin synthesis, transcription factors, pattern-recognition receptors, and pathogenesis-related proteins may regulate kiwifruit resistance to B. dothidea. DEGs involved in calcium signaling, MAPK, and cell wall modification in the resistant variety were induced at an earlier stage and at higher levels compared with the susceptible variety. Thirty DEGs involved in plant defense response were strongly induced in the resistant variety at all three time points. This study allowed the first comprehensive understanding of kiwifruit transcriptome in response to B. dothidea and may help identify key genes required for ripe rot resistance in kiwifruit.

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Unfunded studies
Enter: The author(s) received no specific funding for this work. Yes -all data are fully available without restriction  biotechnology is considered to be one of the most effective management strategies. 59 Studies on the molecular mechanisms of resistance to kiwifruit ripe rot are limited,    In the present study, we explored the defense response of a susceptible kiwifruit 85 genotype ("Hongyang", HY) and a resistant genotype ("Jinyan", JY) infected by B.   Kiwifruits hanging on the tree were surface sterilized with 75% ethanol, peels were air-102 dried, and epidermal tissue of 5 mm in diameter was removed. This was then inoculated 103 with a mycelial disc of 5 mm diameter and covered with moist cotton. In parallel, 104 control fruits were inoculated with sterile agar discs. All treated and control fruits were 105 covered with plastic bags to maintain humidity. Three biological replicates were 106 maintained for each treatment, three fruits were pooled together for each replicate. implemented using GOseq R package [23] in which gene length bias was corrected.

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GO terms with corrected P-values < 0.05 were considered significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) pathway 155 analysis of differentially expressed genes was performed by KOBAS software [24].    and approximately 5% of the genes had FPKM ≥ 60 in each library (S2 Table). The 213 high correlation coefficient of the three biological replicates assured that the analysis 214 was reliable (Fig 1C).  Table). In R, 579 (352 up-regulated; 227 down-regulated), at all three stages of infection (Fig 2A, S4 Fig) Table).   Table). Four GO terms associated with oxidation-reduction were found (GO: 0055114,  Table). There was no GO term associated with 266 sustained down-regulated genes at the three time points in R or S. Some GO terms (S4 267   Table; GO: 0016762 and GO: 0048046) up-regulated in the R variety were down-268 regulated in the S variety. Their difference in expression indicated a potential role in 269 regulating the resistance mechanism to B. dothidea in R.

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To further identify the biological pathways in which the DEGs were involved, we 271 performed KEGG analysis. In total, 6,557 DEGs were assigned to 113 KEGG pathways 272 and 35 of them were significantly enriched (P-value＜0.05). The "metabolic pathway" 273 (223, 1.61 %) was the most significant enriched term, followed by "protein processing" 274 (136, 0.98%) and "carbon metabolism" (124, 0. 89 %) (S5 Table). Comparing treated 275 samples with control samples in both varieties, 24 pathways were significantly up-276 regulated and 11 pathways were significantly down-regulated (S5 Table). KEGG   Table). Comparing R genes was not much lower (Fig 3). Collectively, 30 suatained up-regulated genes 305 encoding PRRs, MAPK, calcium signaling, TFs, hormone metabolism, and cell wall 306 modification-related genes were identified to play role in kiwifruit resistance to B. 307 dothidea (Fig 4). To confirm the accuracy of RNA-seq data, eight co-expressed DEGs involved in 324 resistant to B. dothidea in R were selected for RT-qPCR analysis (Achn251121, 325 Achn104901, Achn040411, Achn012851, Achn386421, Achn372801, Achn315051, 326 Achn327381). Analysis showed the same expression of up-regulation or down-327 regulation as RNA-seq (Fig 5), however, the degree of expression varies between the 328 two data sets beacause of the different sensitivity of Illumina sequencing and RT-qPCR.

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In general, these results suggested the reliability of RNA-seq to analyse the  As far as we know, this is the first report to use RNA-seq technology to identify the 340 genes of resistant and susceptible kiwifruit varieties in response to B. dothidea invasion.

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A total of 13,898 DEGs were detected between "Jinyan" and "Hongyang" and 2,373 342 potential defense-related genes involved in PTI and ETI were identified by searching 343 the keywords in the gene annotation, determined by a manual literature search (S6 Table) 344 and are discussed below. 346 In the present study, there was no significant difference in the expression levels of PRRs 347 genes in R genotype compared with in S genotype at the first time point (1st day after 348 incubation), however, the expression levels in R were higher than in S (S6 Table;  Botrytis cinerea infection and regulated phytoalexin production [32]. Taken  Novel00884 (CDPK), and Achn199221 (CaMBP) were up-regulated in R genotype but 387 were down-regulated in S genotype (S6 Table). Three transcripts encoding CaM or 388 CML (Achn089411, Achn201941, and Achn327381), two transcripts encoding 389 CaMBPs (Achn012851 and Achn124411), and two transcripts encoding other Ca 2+ -390 binding proteins (Achn017121 and Achn024491) (S6 Table, Fig 4) showed sustained,  [47,48].

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In this study, expression of PP2C genes in R occurred earlier than those in S, five PP2C 434 DEGs (Achn286741, Novel04947, Achn194931, Achn104901, and Achn251121) were 435 detected in the study and showed different expression patterns in both varieties.

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Achn286741 and Novel04947 were up-regulated in R but down-regulated in S; 437 Achn194931, Achn104901, and Achn251121 were sustained up-regulated at all three 438 time points in R and only at the third time point in S (S6 Table, Fig 4) Table, Fig 4).  Table, Fig 4) overexpressing the extensin gene limits Pseudomonas syringae invasiveness [58]. 472 Similarly, the mutation in bc1 encoding a COBRA protein caused cell wall thinness 473 and reduction in cellulose content in rice [59]. We detected that five DEGs (S6 Table,   Overall, the mechanism of plant defense response against B. dothidea is complex. DEGs involved in PRRs, MAPK signaling, calcium signaling, Hormone metabolism 509 pathways, TFs pathways and cell wall modification ,which were reported previously as 510 relevant to defense response, were explored, and 30 candidate genes relate to plant 511 defense response were identified from these pathways for future study. We propose a 512 putative network underlying the sustained expression of defense-related DEGs in 513 "Jinyan" (Fig 6). PRR proteins were activated by some effector proteins, leading to 514 activation of MAPK signaling or calcium signaling. Hormone metabolism and TFs 515 pathways were also activated, which trigger defense responses like cell wall 516 modification, stomatal closure, and phytoalexin generation. This study provides a better