Graft incompatibility between pepper and tomato elicits an immune response and triggers localized cell death

Abstract

Graft compatibility is the capacity of two plants to form cohesive vascular connections. Tomato and pepper are incompatible graft partners; however, the underlying cause of graft rejection between these two species remains unknown. We diagnosed graft incompatibility between tomato and diverse pepper varieties based on weakened biophysical stability, decreased growth, and persistent cell death using viability stains. Transcriptomic analysis of the junction was performed using RNA sequencing, and molecular signatures for incompatible graft response were characterized based on meta-transcriptomic comparisons with other biotic processes. We show that tomato is broadly incompatible with diverse pepper cultivars. These incompatible graft partners activate prolonged transcriptional changes that are highly enriched for defense processes. Amongst these processes was broad nucleotide-binding and leucine-rich repeat receptors (NLR) upregulation and genetic signatures indicative of an immune response. Using transcriptomic datasets for a variety of biotic stress treatments, we identified a significant overlap in the genetic profile of incompatible grafting and plant parasitism. In addition, we found over 1000 genes that are uniquely upregulated in incompatible grafts. Based on NLR overactivity, DNA damage, and prolonged cell death, we hypothesize that tomato and pepper graft incompatibility is characterized by an immune response that triggers cell death which interferes with junction formation.

Figure 1

Heterografted pepper fails to form vascular connections and shows a significant decrease in size 30 DAG. (a, c, e, g, i, k, m, o, q, s, u, w, y) Representative photographs and (b, d, f, h, j, l, n, p, r, t, v, x, z) confocal micrographs for self-grafted tomato (a, b), self-grafted habanero (c, d), tomato:Habanero (e, f), Habanero:tomato (g, h), self-grafted Doux des Landes (DDL) (i, j), tomato:DDL (k, l), DDL:tomato (m, n). self-grafted Cayenne (o, p), tomato:Cayenne (q, r), Cayenne:tomato (s, t), self-grafted California Wonder (CW) (u, v), tomato:CW (w, x), CW:tomato (y, z). Graft junctions were stained with propidium iodide and imaged on a confocal microscope. Pink arrows indicate a successful graft junction with a healed xylem (b, d, j, p, v, aa). White arrows indicate a failed vascular reconnection (f, h, l, n, r, t, x, z, ab) and white asterisk highlight adventitious roots (f, n, t). All plant image scale bars are 5 cm, and all micrograph scale bars are 1000 μm. (aa) A zoomed up view of self-grafted tomato to demonstrate successful xylem connections. (ab) A zoomed view of heterograft tomato:cayenne junction to demonstrate failed xylem connections. Blue arrowheads denote xylem in the graft junction of aa and ab.

Figure 2

Incompatible grafts contain persistent NVT over time. (a–r) Representative images of 2.5 mm long graft junctions at 7, 14, and 21 DAG stained with Trypan Blue. A representative ungrafted tomato stem and the percent of NVT are shown at 7 DAG (a, s), 14 DAG (g, y), and 21 DAG (m, ae). A representative ungrafted pepper stem and the percent of NVT at 7 DAG (b, t), 14 DAG (h, z), and 21 DAG (n, af). A representative self-graft tomato junction and the percent of NVT at 7 DAG (c, u), 14 DAG (i, aa), and 21 DAG (o, ag). A representative self-grafted pepper junction and the percent of NVT at 7 DAG (d, v), 14 DAG (j, ab), and 21 DAG (p, ah). A representative tomato:pepper junction and the percent of NVT at 7 DAG (e, w), 14 DAG (k, ac), and 21 DAG (q, aI). A representative pepper:tomato junction and the percent of NVT at 7 DAG (f, x), 14 DAG (l, ad), and 21 DAG (r, aj). Yellow arrows point to examples of tissue death; dashed lines signify the graft site; all junctions are 2.5 mm tall. All images set to same scale, with the scale bar equal to 1 cm (a–r). (s–aj) The percent of cell death and (ak) the area of cell death in the junction of all graft combinations at 7, 14, and 21 DAG. From left to right, dark orange depicts ungrafted tomato, light orange depicts ungrafted pepper, green depicts self-grafted tomato, teal depicts self-grafted pepper, purple depicts tomato:pepper, and pink depicts pepper:tomato. Compact letter display based on adjusted p-value of Tukey’s HSD test. Biological replicates are depicted as jitter (ak) as well as described in detail in Table S4.

Figure 3

Incompatible heterografts have prolonged differential gene regulation compared to self-grafts. Differentially expressed genes (|1.5| , adjusted p-value<0.05) of each grafted tissue (compared to ungrafted) at each time point for tomato and pepper. Upregulated genes are shown in light colors and downregulated genes are shown in dark colors. Self-grafted scions are dark purple, self-grated stocks are light purple, heterografted scions are orange, and heterograft stocks are yellow. Each combination has 3–5 bio-replicates.

Figure 4

Incompatible graft-specific upregulated genes are involved in defense response. (a, b) Uniquely upregulated incompatible graft genes were determined by performing likelihood ratio testing (p < 0.05) on ungrafted, self-graft scion, and incompatible graft scion as well as ungrafted, self-grafted stock, and incompatible stock tissue. The genes upregulated in only the incompatible graft tissue were used to perform GO enrichment. GO terms enriched in incompatible grafted tomato tissue at 7, 14, and 21 DAG (a). GO terms enriched in incompatible grafted pepper tissue at 7, 14, and 21 DAG (b). (c, d) Log-fold change of NLRs in grafted tissue compared to ungrafted tissue of tomato (c) and pepper (d). (e) The log-fold change of genes involved in hypersensitive response in grafted vs. ungrafted tissue. The log-fold change was scaled by row. The tissue is denoted by the colored columns, from left to right, where self-grafted scions are dark purple, self-grafted stocks are light purple, incompatible grafted scions are orange, and incompatible grafted stocks are yellow. The days after grafting were denoted by colored columns, from left to right, where 7 DAG are white, 14 DAG are grey, and 21 DAG are black. Astrix denotes adjusted P-value<0.05 and log-fold change greater than |1.5|.

Figure 5

The steroidal glycoalkaloid biosynthesis pathway among significantly upregulated processes in heterografted grafted scions. (a) Upregulated genes for all graft combinations were determined in comparison to ungrafted stems, from left to right, at 7 (light grey), 14 (dark grey), and 21 (black) DAG in the self-grafted scion (dark purple), self-grated stock (magenta), heterografted scion (orange), and heterografted stock (yellow). Significant differential expression qualified by LFC greater than |1.5| and adjusted p-value <0.05. Significance is denoted with an asterisk. (b) Orthologs upregulated at any given tissue/time point in both tomato and pepper. Orthogroups were determined between Solanum lycopersicum, Capsicum annum, and Arabidopsis thaliana using OrthoFinder. Upregulated genes for all graft combinations were determined in comparison to ungrafted stems. Each gene had a corresponding orthogroup. A shared ortholog was determined if upregulated genes (lfc >1.5, adjusted <0.05) from both tomato and pepper at a common tissue/time point were linked to the same orthogroup. (c) Normalized read counts of SlERF114 and CaERF114 were across time. Read counts for tomato and pepper were normalized and faceted by tissue type. Box plots of ungrafted tissue read counts are orange, boxplots of scions tissue read counts are purple, and boxplots of stock tissue read counts are pink.

Figure 6

Grafting elicits unique an shared genetic processes with other biological stressors. (a) Spearman Rank Correlation between 7 DAG samples, botrytis infection, herbivory, plant parasitism, and arbuscular mycorrhizal fungi (AMF) colonization. (b) Overlap of upregulated genes from four biological processes investigated. (c) Upset plot showing the overlap between upregulated genes from the biological processes: AMF, plant parasitism, insect herbivory, and fungal infection, scion or stock self-, and scion or stock incompatible graft tissue 7 DAG. (d–g) Overlap of upregulated genes from scion or stock of self- or incompatible-grafted tissue at 7 DAG and all biological processes. (d) The overlap between grafting and AMF, (e) botrytis fungal infection, (f) herbivory, (g) and plant parasitism. The grey outline denotes the biological stressors, whereas AMF was used as a control.