Dual RNA-Seq Analysis of the Pine- Fusarium circinatum Interaction in Resistant (Pinus tecunumanii) and Susceptible (Pinus patula) Hosts

Abstract

Fusarium circinatum poses a serious threat to many pine species in both commercial and natural pine forests. Knowledge regarding the molecular basis of pine-F. circinatum host-pathogen interactions could assist efforts to produce more resistant planting stock. This study aimed to identify molecular responses underlying resistance against F. circinatum. A dual RNA-seq approach was used to investigate host and pathogen expression in F. circinatum challenged Pinus tecunumanii (resistant) and Pinus patula (susceptible), at three- and seven-days post inoculation. RNA-seq reads were mapped to combined host-pathogen references for both pine species to identify differentially expressed genes (DEGs). F. circinatum genes expressed during infection showed decreased ergosterol biosynthesis in P. tecunumanii relative to P. patula. For P. tecunumanii, enriched gene ontologies and DEGs indicated roles for auxin-, ethylene-, jasmonate- and salicylate-mediated phytohormone signalling. Correspondingly, key phytohormone signaling components were down-regulated in P. patula. Key F. circinatum ergosterol biosynthesis genes were expressed at lower levels during infection of the resistant relative to the susceptible host. This study further suggests that coordination of phytohormone signaling is required for F. circinatum resistance in P. tecunumanii, while a comparatively delayed response and impaired phytohormone signaling contributes to susceptibility in P. patula.

Keywords: Dual RNA-seq; Fusarium circinatum; Pinus patula; Pinus tecunumanii; ergosterol biosynthesis; host-pathogen interaction.

Figure 1

Expression of Fusarium circinatum ergosterol biosynthesis genes during infection. The y-axes represent average FPKM across biological replicates for inoculated samples from P. patula (blue) and P. tecunumanii (green) at 3- (dark colours) and 7- (light colours) dpi. Error bars represent the standard error of the mean (n = 3). Letters above bars indicate significant difference in expression (FDR < 0.05). Dashed black outlines indicate high-confidence expressed genes. ERG10 = acetyl-CoA acetyltransferase, HMG1 = 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, ERG12 = mevalonate kinase, ERG8 = phosphomevalonate kinase, MVD1 = mevalonate pyrophosphate decarboxylase, IDI1 = isopentenyl diphosphate:dimethylallyl diphosphate isomerase, ERG20 = geranylgeranyl diphosphate synthase, ERG9 = farnesyl-diphosphate farnesyltransferase, ERG1 = squalene epoxidase, ERG7 = lanosterol synthase, ERG11 = CYP51/sterol-14α-demethylase, ERG24 = δ14-sterol reductase, ERG25 = C-4 methylsterol oxidase, ERG26 = sterol-4α-carboxylate 3-hydrogenase, ERG27 = 3-keto steroid reductase, ERG6 = sterol 24-C-methyltransferase, ERG2 = C-8 sterol isomerase, ERG3 = δ7-sterol 5-desaturase, ERG5 = CYP61a/sterol 22-desaturase, ERG4 = δ24(24(1))-sterol reductase. ERG13 (HMG-CoA synthase) was absent from the transcriptome.

Figure 2

Summary of phytohormone related host DEGs during F. circinatum challenge. Up- and down-regulation (inoculated relative to mock-inoculated) of genes related to cytokinin, abscisic acid, gibberellic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and auxin signalling, in P. patula (blue) and P. tecunumanii (green) at three (dark colours) and seven (light colours) days post inoculation, are indicated by arrows (↑ and ↓ respectively; Additional file 5: Tables S4 and S5). Dotted red lines indicate suppression, dashed black lines indicate positive interaction, solid black lines indicate enzymatic reactions. Borderless text indicates processes, square bordered text indicates proteins, round bordered text indicates compounds. AAO3—abscisic-aldehyde oxidase; A-ARR—type A Arabidopsis response regulator; ABA—abscisic acid; ABF—ABA response factor; ABI—ABA insensitive; ACAA1—acetyl-Coenzyme A acyltransferase 1; ACC—1-aminocyclopropane-1-carboxylic acid; ACO—ACC oxidase; ACS—ACC 1-aminocyclopropane-1-carboxylic acid synthase; AHP—Arabidopsis histidine phosphotransferase; Ala—alanine; AMY—α-amylases; AOC—allene oxide cyclase; AOS—allene oxide synthase; ARF—auxin response factor; Asp—aspartic acid; Aux/IAA—auxin inhibitor; AUX/LAX—auxin influx carriers; BAK1—BRI1-associated kinase; B-ARR—type B Arabidopsis response regulator; BIN2—BR insensitive 2; BR—brassinosteroid; BRH1—brassinosteroid-responsive RING H2; BRI1—brassinosteroid insensitive 1; BSK—BR-signalling kinase; BSU1—protein phosphatase BRI1 suppressor; CAS1—cycloartenol synthase; CDG1—constitutive differential growth 1; CHI—chalcone-flavone isomerase; CHS—chalcone synthase; CIPK—Cbl-interacting protein kinase; CK—cytokinin; CKX—CK dehydrogenase; COI1—coronatine insensitive 1; CPS—ent-copalyl diphosphate synthase; CRF—CK response factor; CTR1—Raf-like ser/thr kinase; CYP—Cytochrome P450 family protein; DOGT1—DON-glucosyltransferase 1; EDS1—enhanced disease susceptibility 1; EIN—ET insensitive; ERF—ET response factor; ERG6—sterol 24-C-methyltransferase; ET—ethylene; ETR1—ET receptor; GA—gibberellic acid; GA2ox—GA 2-oxidase; GA3ox—GA 3-oxidase; GAMT—GA methyl transferase; GH3—Gretchen Hagen3 family protein; GID1—GA insensitive dwarf 1; Glu—glutamine; GST—glutathione-S-transferase; HK—histidine kinase; IAA—indole-3-acetic acid; IAMT—IAA methyl transferase; ILL—JA-Ile/IAA-amino acid hydrolase; IPT2—isopentenyl transferase; JA—jasmonic acid; JA-Ile—jasmonoyl-isoleucine; JAR1—JA-amino acid synthetase; JAZ—jasmonate zim-domain family transcription repressor proteins; JMT—JA methyl transferase; LBD—lob domain-containing protein; Leu—leucine; LFY—LEAFY; LOX—lipoxygenase; MeJA—methyl-jasmonate; MES—methyl esterase; MeSA—methyl-salicylate; MFP2—multifunctional protein 2; MYC—JA responsive transcriptome factor; NCED—9-cis-epoxycarotenoid dioxygenase; NINJA—novel interactor of JAZ; NPR—non-expressor of PR; NSY—neoxanthin synthase; OPCL—OPC8-CoA ligase; OPR—12-oxophytodienoate reductase; PAD4—phytoalexin deficient 4; PIF3—phytochrome-interacting factor 3; PIN—auxin efflux transporter; PP2C—type 2C protein phosphatases; PR—pathogenesis related proteins; PYR/PYL—ABA receptors; RTE1—reversion-to-ethylene sensitivity 1; SA—salicylic acid; SAM—S-adenosyl-methionine synthetase; SAMT—SA methyl transferase; SAUR—small auxin up RNA protein; SCF—Skp-cullin-F-box complex; SDR—xanthoxin dehydrogenase; SMT2—24-methylenesterol C-methyltransferase; SOD—superoxide dismutase; TCH4—Xyloglucan endotransglucosylase hydrolase protein; TGA—TGA family transcription factors; TPL—topless; Ub—ubiquitin; UGT—UDP-Glycosyl/Glucosyl/Glucuronosyl transferase; UMP—ubiquitin mediated proteolysis; ZEP—zeaxanthin epoxidase.