Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection

Abstract

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Keywords: Carya; DEG; Fungal resistance; Pecan; Provenance; RNA-seq; Scab; Venturia.

Fig. 1

Boxplots of RNAseq data mapped to either the Carya illinoinensis transcriptome (A) or Venturia effusa genome (B). RNAseq data was generated from susceptible non-diseased leaves (S-ND), diseased susceptible leaves (S-D), and resistant leaves (R). The double asterisk (**) indicates a Tukey’s HSD p-value < 0.05 for a given treatment. Reads mapped to the high expressing, non-orthologous Venturia effusa elongation factor one-alpha are shown (C). Whiskers represent the minimum of either 1.5 times the interquartile range or the extreme of the sample range. Open circles indicate outliers

Fig. 2

Quantitative PCR showing relative expression of two genes in pecan leaves collected in 2023 (n=4) using polyadenylate-binding protein (Caril.16G041200) as a reference gene. Expression patterns were similar to DEGs identified in RNAseq data from samples collected in 2019, showing up-regulation of a cystine-rich receptor-like kinase (Caril.09G152000, A) in resistant leaves (R) compared to susceptible non-diseased leaves (S-ND), while myrcene synthase (Caril.05G234200, B) was down-regulated in resistant leaves (R)

Fig. 3

Venn diagram showing number and fold-change type of differentially expressed genes (DEG) for non-diseased susceptible pecan leaves (S-ND) when compared separately to diseased (S-D) and resistant (R) leaves. Treatments compared are above their respective results. Up- (Up) or down-regulation (Dn) describes expression pattern of underlined treatment group, when compared to S-ND. Fold-change patterns for DEGs shared between comparisons is described in detail below Venn diagram with arrows indicating direction of increasing expression

Fig. 4

Differentially expressed predicted R-genes for pecan scab related treatments. Regulation type explains fold change for (A) resistant leaves (R), and (B) diseased leaves (S-D) when compared to susceptible non-diseased leaves (S-ND). Abbreviations: N=nucleotide binding site (NBS), L=leucine rich-repeat (LRR), NL=NBS-LRR, TNL=TIR-NB-LRR, T=toll-interleukin receptor (TIR), CN=coiled-coil (CC)-NBS, CK=CC-kinase, CL=CC-LRR, CLK=CC-LRR-kinase, KIN=kinase, RLK=receptor-like kinase, RLP=receptor like protein

Fig. 5

Differentially expressed (DE) transcription factors (TFs) for different pecan scab treatments. Regulation type explains fold change for (A) resistant leaves (R), and (B) susceptible diseased leaves (S-D) when compared to non-diseased susceptible leaves (S-ND). Line graph shows number of expected DE TFs if chosen at random from all Carya illinoinensis TFs as predicted with PlantTFDB v5.0 (1/2 DE TF * ratio C. illinoinensis TF class)

Fig. 6

Differentially expressed genes (DEGs) in the “plant-pathogen interaction” KEGG pathway 04626 for (A) pecan (Carya illinoinensis) trees resistant to Venturia effusa (R) as opposed to susceptible non-diseased trees (S-ND) and (B) susceptible diseased leaves (S-D) compared to susceptible non-diseased (S-ND) leaves [48]. Significant DEGs have a Benjamini & Hochberg adjusted p-value < 0.05 and an absolute log2 fold change > 1.5. Genes in the figure with no expression information are not differentially expressed to a significant extent or C. illinoinensis DEGs have no Arabidopsis thaliana ortholog information. Pathway element in the figure shows expression pattern for each individual in the case group (R or S-D) compared to S-ND

Fig. 7

Gene expression at predicted susceptibility quantitative trait loci (QTL) and flanking regions in scab resistant leaves of pecan (Carya illinoinensis 87MX3-2.11). Red gene names indicate genes that are significantly down-regulated. Eight of the nine differentially expressed genes (DEGs) are predicted leucine rich repeat R-genes. Expression of most proximal 5’ and 3’ genes also shown