Comparative transcriptome analysis of a lowly virulent strain of Erwinia amylovora in shoots of two apple cultivars - susceptible and resistant to fire blight

Abstract

Background: Erwinia amylovora is generally considered to be a homogeneous species in terms of phenotypic and genetic features. However, strains show variation in their virulence, particularly on hosts with different susceptibility to fire blight. We applied the RNA-seq technique to elucidate transcriptome-level changes of the lowly virulent E. amylovora 650 strain during infection of shoots of susceptible (Idared) and resistant (Free Redstar) apple cultivars.

Results: The highest number of differentially expressed E. amylovora genes between the two apple genotypes was observed at 24 h after inoculation. Six days after inoculation, only a few bacterial genes were differentially expressed in the susceptible and resistant apple cultivars. The analysis of differentially expressed gene functions showed that generally, higher expression of genes related to stress response and defence against toxic compounds was observed in Free Redstar. Also in this cultivar, higher expression of flagellar genes (FlaI), which are recognized as PAMP (pathogen-associated molecular pattern) by the innate immune systems of plants, was noted. Additionally, several genes that have not yet been proven to play a role in the pathogenic abilities of E. amylovora were found to be differentially expressed in the two apple cultivars.

Conclusions: This RNA-seq analysis generated a novel dataset describing the transcriptional response of the lowly virulent strain of E. amylovora in susceptible and resistant apple cultivar. Most genes were regulated in the same way in both apple cultivars, but there were also some cultivar-specific responses suggesting that the environment in Free Redstar is more stressful for bacteria what can be the reason of their inability to infect of this cultivar. Among genes with the highest fold change in expression between experimental combinations or with the highest transcript abundance, there are many genes without ascribed functions, which have never been tested for their role in pathogenicity. Overall, this study provides the first transcriptional profile by RNA-seq of E. amylovora during infection of a host plant and insights into the transcriptional response of this pathogen in the environments of susceptible and resistant apple plants.

Keywords: Fire blight; RNA-seq; Virulence.

Fig. 1

Mean virulence rating of E. amylovora 650 strain used for inoculation of actively growing shoots of three apple cultivars of different susceptibility. Virulence was measured 6 weeks post-inoculation and it is expressed as a percent of the length of a shoot exhibiting necrosis divided by the entire length of shoot. Mean virulence ratings were separated with Tukey’s test at a significance level of P = 0.05. The vertical bars represent standard error

Fig. 2

The Principal Component Analysis (PCA) of the log₂ - transformed normalized expression values highlighted the variability between the samples. FR – Free Redstar, I – Idared, 24 h – 24 h after inoculation, 6 days – 6 days after inoculation

Fig. 3

Validation of RNA-seq data using RT-qPCR. Fold changes of gene expression detected by RNA-seq were plotted against the data of qPCR. The reference line indicates the linear relationship between the results of RNA-seq and qPCR

Fig. 4

Venn diagram representing number of E. amylovora genes a/ up- and b/ down-regulated in planta comparing to expression of genes of pure bacterial culture grown overnight in TY medium. I – Idared; FR – Free Redstar; 24 h – sample collected 24 h after inoculation; 6d - sample collected 6 days after inoculation

Fig. 5

Relative abundance of CDSs assigned to each eggNOG functional categories for commonly a/ 640 down and b/ 698 up-regulated E. amylovora genes in planta. The eggNOG functional categories are as follows: C, energy production and conversion; D, cell cycle control, cell division and chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation; K, transcription; L, replication; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking and secretion; V, defence mechanisms. ↑- over-represented; ↓ - under-represented COG categories calculated based on hypergeometric distribution at FDR < 0.05

Fig. 6

Diagram showing the study design. One-year-old, potted apple trees cultivars Idared and Free Redstar grafted on M.26 were inoculated with E. amylovora strain 650 in greenhouse conditions. Inoculation was made on actively growing shoots, with bacterial suspension grown overnight in TY medium. After 24 h and 6 days from the inoculation time total RNA was isolated from inoculated shoots. At each time point, RNA was isolated separately from at least 6 shoots of each apple cultivar. Additionally, RNA was isolated from the pure culture of E. amylovora 650 grown overnight in TY medium. In each time point, RNA was isolated from 3 biological replicates (in case of pure bacterial culture - two biological replicates). The replicates constituted the sample (marked by red circle)