Abstract

   Herbaceous peony (Paeonia lactiflora Pall.) is a well-known traditional
   flower in China and is widely used for landscaping and garden greening
   due to its high ornamental value. However, disease spots usually appear
   after the flowering of the plant and may result in the withering of the
   plant in severe cases. This study examined the disease incidence in an
   herbaceous peony field in the Yangzhou region, Jiangsu Province. Based
   on morphological characteristics and molecular data, the disease in
   this area was identified as a gray mold caused by Botrytis cinerea.
   Based on previously obtained transcriptome data, eight libraries
   generated from two herbaceous peony cultivars ‘Zifengyu’ and ‘Dafugui’
   with different susceptibilities to the disease were then analyzed using
   digital gene expression profiling (DGE). Thousands of differentially
   expressed genes (DEGs) were screened by comparing the eight samples,
   and these genes were annotated using the Gene ontology (GO) and Kyoto
   encyclopedia of genes and genomes (KEGG) database. The pathways related
   to plant-pathogen interaction, secondary metabolism synthesis and
   antioxidant system were concentrated, and 51, 76, and 13 disease
   resistance-relevant candidate genes were identified, respectively. The
   expression patterns of these candidate genes differed between the two
   cultivars: their expression of the disease-resistant cultivar
   ‘Zifengyu’ sharply increased during the early stages of infection,
   while it was relatively subdued in the disease-sensitive cultivar
   ‘Dafugui’. A selection of ten candidate genes was evaluated by
   quantitative real-time PCR (qRT-PCR) to validate the DGE data. These
   results revealed the transcriptional changes that took place during the
   interaction of herbaceous peony with B. cinerea, providing insight into
   the molecular mechanisms of host resistance to gray mold.

Introduction

   Botrytis cinerea Pers. (teleomorph: Botryotinia fuckeliana (de Bary)
   Fuck.), which leads to gray mold on various host plants [[33]1], is
   considered one of the most important fungal plant pathogens worldwide
   [[34]2]. As a necrotrophic fungus, it grows by relying on the nutrition
   of necrotic tissue after infecting hosts to trigger a hypersensitive
   response (HR); it always interferes with the physiological and
   biochemical functions of plants and may even cause the plants to wither
   and die [[35]3–[36]6]. Plants evolve several mechanisms to cope with B.
   cinerea stress, and these mechanisms are all achieved by induction of
   numerous disease resistance genes involved in various pathways
   [[37]5–[38]8]. A WRKY family gene that responds to B. cinerea
   infection, (Solanum lycopersicum defense-related WRKY1) SlDRW1, is
   significantly up-regulated by the defense response of tomato, and the
   silencing of this gene increases the severity of gray mold [[39]8]. De
   Cremer et al. [[40]6] show that genes involved in the phenylpropanoid
   pathway and terpenoid synthesis are transcribed in lettuce in response
   to a challenge by B. cinerea. The above studies reflect that disease
   resistance-relevant genes are highly effective against B. cinerea in
   host plants.

   The continuous improvement of RNA-seq technology has provided a new
   approach for the functional genomics study of plants at the
   transcriptome level [[41]9]. Digital gene expression profiling (DGE) is
   a new approach for transcriptome analysis that integrates
   high-throughput sequencing technology and high-performance computing
   analysis technology. It is mainly used to quantitatively study the gene
   expression profiles of specific tissues or cells in one species during
   specific biological processes, particularly concentrating on the study
   of gene expression differences at genome-wide level [[42]10, [43]11].
   DGE technology has lots of advantages, such as more accurate
   quantification, higher repeatability, wider detection range, and more
   reliable analysis. Recently, RNA-Seq has been widely used to study
   plants. However, few studies have examined the molecular mechanism of
   plant disease resistance via RNA-Seq and DGE technology. The use of DGE
   technology identifies numerous candidate genes that are specifically or
   commonly regulated at different stages of HR in an analysis of
   Chenopodium amaranticolor inoculated with Tobacco mosaic virus and
   Cucumber mosaic virus, highlighting the dynamic changes of the
   differentially expressed genes (DEGs) in the plant-pathogen interaction
   pathway [[44]12]. DGE analysis is also used to compare healthy and
   infected tobacco plants at six sequential disease developmental stages
   and to identify thousands of DEGs and many biological processes
   involved in disease resistance response [[45]13]. Analogously, Sun et
   al. [[46]14] compared upland cotton and sea-island cotton infected with
   Verticillium dahliae and found that the hydroxycinnamoyl transferase
   gene (HCT) is up-regulated in upland cotton, whereas Phenylalanine
   Ammonialyase gene (PAL), 4-Coumarate:CoA ligase gene (4CL), Cinnamoyl
   Alcohol Dehydrogenase gene (CAD), Caffeoyl-CoA5-O-methyltransfenase
   gene (CCoAOMT), and caffeicacid-5-O-methyltransfenase gene (COMT) are
   up-regulated in sea-island cotton in the phenylalanine metabolism
   pathway. The successful uses of DGE technology described above provide
   a reference for studies of the molecular mechanism of other plants in
   response to B. cinerea infection.

   Herbaceous peony (Paeonia lactiflora Pall.) is a well-known traditional
   flower in China with a reputation as “the minister of flowers”. It is
   widely used in landscaping and garden greening due to its large flower,
   elegant shape, gorgeous color and rich fragrance, and it has been
   extensively cultivated in more than 50 countries, such as the United
   States, France, the Netherlands, etc [[47]15]. However, gray mold
   invariably develops on herbaceous peony plants grown in the Jiangsu and
   Zhejiang area of China, as evidenced by wizened and necrotic leaves,
   sunken and broken stems and brown and rotten petals [[48]16]. These
   symptoms seriously affect the ornamental and commercial values of the
   plants. This study revealed that the herbaceous peony cultivars
   ‘Zifengyu’ and ‘Dafugui’ had been damaged by gray mold but that their
   resistance to the pathogen differed significantly. ‘Zifengyu’ grew well
   and showed few disease spots, while ‘Dafugui’ appeared weak and almost
   completely withered. The pathogen was identified as B. cinerea in both
   cases based on morphological characteristics and molecular data. At
   present, studies of the defense mechanism of herbaceous peony against
   B. cinerea are scarce and have mostly concentrated on the physiological
   and biochemical aspects of pathogenic fungus identification, biological
   characteristics, screening of resistant cultivars and chemical control
   [[49]16]. Little is known about the disease resistance-relevant genes
   involved in the interaction between host and pathogen, which requires
   exploration into these mechanisms. In a previous study, transcriptome
   sequencing of leaves of two herbaceous peony cultivars ‘Zifengyu’ and
   ‘Dafugui’ harvested at four stages (from uninfected to severely
   infected, at May 25, June 15, July 5, and July 25, respectively) were
   performed via de novo RNA-seq technology to establish a database
   (Accession No. for library ‘Zifengyu’ SRS774325; Accession No. for
   library ‘Dafugui’ SRS774327). Based on this database, a DGE analysis of
   these two cultivars samples was conducted at four stages (the same as
   the transcriptome sequencing, from uninfected to severely infected, at
   May 25, June 15, July 5, and July 25, respectively) in order to
   identify the metabolic pathways and disease resistance-relevant genes
   of herbaceous peony plants that were involved in B. cinerea infection.
   These pathways and genes could provide a theoretical basis for
   comprehensively expounding the mechanism of herbaceous peony resistance
   to gray mold.

Materials and Methods

Plant materials

   The herbaceous peony disease-resistant cultivar ‘Zifengyu’ and the
   disease-sensitive cultivar ‘Dafugui’ were examined in this study, which
   were planted in the germplasm repository of Horticulture and Plant
   Protection College, Yangzhou University, Jiangsu Province, P. R. China
   (32°30′N, 119°25′E). Because the disease severity gradually increased
   after flowering, the third and fourth node leaves at the top of the
   branch were taken at four stages from uninfected to severely infected:
   Stage 1 (S1, May 25), Stage 2 (S2, June 15), Stage 3 (S3, July 5),
   Stage 4 (S4, July 25); and S1 was the uninfected stage that taken as
   the control. One part of samples was used to identify the pathogenic
   fungus and determine physiological indexes, while the remainder was
   stored at -80°C in order to extract RNA for DGE and quantitative
   real-time PCR (qRT-PCR) analysis.

Pathogen identification

   The pathogenic fungus was isolated and purified via usual tissue
   isolation methods [[50]17], and then isolated strains were cultured on
   potato sugar agar (PSA) plate medium to observe the colony morphology.
   After sporulation, the morphological characteristics were observed,
   photomicrographs of conidiophores and conidia were obtained, and the
   conidia size was measured. The healthy leaves were then inoculated in
   vivo and in vitro with the dominant pathogen with or without wounds,
   respectively. The tissue was again harvested after infection to isolate
   the pathogen from the leaves and verify that whether this pathogen was
   the same as the original inoculant. The pathogen morphology was
   preliminarily identified based on the references [[51]18]. The genomic