Effects of 'Candidatus Liberibacter solanacearum' haplotypes A and B on tomato gene expression and geotropism

Abstract

Background: The tomato psyllid, Bactericera cockerelli Šulc (Hemiptera: Triozidae), is a pest of solanaceous crops such as tomato (Solanum lycopersicum L.) in the U.S. and vectors the disease-causing pathogen 'Candidatus Liberibacter solanacearum' (or Lso). Disease symptom severity is dependent on Lso haplotype: tomato plants infected with Lso haplotype B experience more severe symptoms and higher mortality compared to plants infected with Lso haplotype A. By characterizing the molecular differences in the tomato plant's responses to Lso haplotypes, the key components of LsoB virulence can be identified and, thus, targeted for disease mitigation strategies.

Results: To characterize the tomato plant genes putatively involved in the differential immune responses to Lso haplotypes A and B, RNA was extracted from tomato 'Moneymaker' leaves 3 weeks after psyllid infestation. Gene expression levels were compared between uninfected tomato plants (i.e., controls and plants infested with Lso-free psyllids) and infected plants (i.e., plants infested with psyllids infected with either Lso haplotype A or Lso haplotype B). Furthermore, expression levels were compared between plants infected with Lso haplotype A and plants infected with Lso haplotype B. A whole transcriptome analysis identified 578 differentially expressed genes (DEGs) between uninfected and infected plants as well as 451 DEGs between LsoA- and LsoB-infected plants. These DEGs were primarily associated with plant defense against abiotic and biotic stressors, growth/development, plant primary metabolism, transport and signaling, and transcription/translation. These gene expression changes suggested that tomato plants traded off plant growth and homeostasis for improved defense against pathogens, especially when infected with LsoB. Consistent with these results, tomato plant growth experiments determined that LsoB-infected plants were significantly stunted and had impaired negative geotropism. However, it appeared that the defense responses mounted by tomatoes were insufficient for overcoming the disease symptoms and mortality caused by LsoB infection, while these defenses could compensate for LsoA infection.

Conclusion: The transcriptomic analysis and growth experiments demonstrated that Lso-infected tomato plants underwent gene expression changes related to abiotic and biotic stressors, impaired growth/development, impaired plant primary metabolism, impaired transport and signaling transduction, and impaired transcription/translation. Furthermore, the transcriptomic analysis also showed that LsoB-infected plants, relative to LsoA-infected, experienced more severe stunting, had improved responses to some stressors and impaired responses to others, had poorer transport and signaling transduction, and had impaired carbohydrate synthesis and photosynthesis.

Keywords: Bactericera cockerelli; Gene expression; Lso haplotype; Plant-insect-microbe interactions; Potato; Psyllid; Solanum lycopersicum L.; Transcriptome; Transcriptomics; zebra chip; ‘Candidatus Liberibacter solanacearum’.

Fig. 1

A Comparative heatmap of relative expression changes among uninfested (Control#), Lso-free psyllid infested (LsoFree#), LsoB-infected (LsoB#), and LsoA-infected (LsoA#) tomato plant DEGs. Dark colors denote down-regulation and light colors denote up-regulation. Lines above the heatmap depict the phylogenetic hiearchy among similar treatments and similar gene expression levels. B Multidimensional scaling (MDS) distance plot of fragments per kilobase per million reads (fpkm) among treatments: Uninfested (Control#), Lso-free psyllid infested (LsoFree#), LsoB-infected (LsoB#), and LsoA-infected (LsoA#)

Fig. 2

g:Profiler analysis of Lso-infected tomato plant DEG homologs depicting their relative overrepresentation among Arabidopsis molecular functions (MF), biological processes (BP), or cellular components (CC). The left axis represents the -log₁₀(p_adj) likelihood that a given MF, BP, or CC would be associated with a random selection of Arabidopsis genes. Circle sizes represent the relative number of times a given MF, BP, CC, or KEGG term appears among analyzed genes. In general, expression changes occurred throughout the cell and were most likely to be involved with response to stress or stimulus, biosynthesis of secondary metabolites, methyltransferases, and DNA binding (likely involved in expression regulation). Labels above, connected to arrows, or adjacent to circles describe specific the MF, BP, or CC associated with each circle; some labels have been omitted due to redundancy

Fig. 3

g:Profiler analysis of Lso haplotype-specific tomato plant DEG homologs depicting their relative overrepresentation among Arabidopsis molecular functions (MF), biological processes (BP), or cellular components (CC). The left axis represents the -log₁₀(p_adj) likelihood that a given MF, BP, or CC would be associated with a random selection of Arabidopsis genes. Circle sizes represent the relative number of times a given MF, BP, CC, or KEGG term appears among analyzed genes. In general, expression changes occurred throughout the cell and were most likely to be involved with response to stress or stimulus, biosynthesis of secondary metabolites, methyltransferases, and DNA binding (likely involved in expression regulation). Labels above, connected to arrows, or adjacent to circles describe specific the MF, BP, or CC associated with each circle; some labels have been omitted due to redundancy

Fig. 4

RT-qPCR results comparing 2^-ΔΔCT values between psyllid treatment groups: Control plants (solid white), Lso-free plants (white with black stripes), LsoB-infected plants (solid black), and LsoA-infected plants (grey with black stripes). Numbers listed above each column represent the average fpkm (fragments per kilobase of transcript per million reads) values calculated for the given treatment and target gene. Target DEGs were selected based on their expected relative expression levels between treatments: Heat shock cognate 70 kDa protein 1 (HSP70–1; Solyc06g076020.3) was expected to be up-regulated in LsoA-infected plants and expressed at even higher levels in LsoB-infected plants, jasmonate ZIM-domain protein 2 was expected to be up-regulated in Lso-infected plants (NtJAZ2; Solyc12g009220.2), ribosomal protein L2 was expected to be down-regulated in infected plants (rL2; Solyc10g047090.2), glutaredoxin-C6 was expected to be down-regulated in infected plants (GRXC6; Solyc11g069940.1), and Ycf1 was expected to be down-regulated in infected plants (Ycf1; Solyc12g035550.1). An asterisk denotes a significant difference with control plants

Fig. 5

Photographs depicting typical growth of tomato plants among psyllid treatment groups: Control plants (A) grew normally and displayed normal negative geotropism, plants infested with Lso-free psyllids (B) were slightly stunted but displayed normal negative geotropism, LsoA-infected plants (C) were significantly stunted and failed to display negative geotropism, and LsoB-infected plants (D) were significantly stunted, failed to display negative geotropism, and would occasionally become limp