Excess salicylic acid mediates graft incompatibility by repressing auxin-dependent vascular reconnection in tomato

Key results

1. 01

   ShownIn compatible self-grafts, SA rises before reconnection and peaks in stocks at 4 DAG in tomato and 6 DAG in pepper. Incompatible heterografts (scion/stock: tomato/pepper T/P, pepper/tomato P/T) accumulate much more SA, mostly in the rootstock, with strong SlPR1 induction.

   At 7 days after grafting (DAG), compatible self-grafts held no more than 36.4 ng/g SA. T/P stocks reached 184.6 ng/g (4.8-fold vs average P/P) and P/T stocks 101.9 ng/g (2.8-fold vs T/T); incompatible scions were similar (ns). pPR1:GUS staining at 4 DAG and SlPR1 RT-qPCR at 7 DAG were higher in heterografts than in tomato self-grafts. · Incompatible rootstocks overaccumulate salicylic…; Figure legends

2. 02

   ShownScions of incompatible T/P grafts are in a primed, hyperactive pattern-triggered immunity (PTI) state and resist bacterial infection better. Grafting alone moderately raises PTI.

   After 100 nM flg22 on scion leaves at 7 DAG, cumulative ROS in T/P was 4 times that of T/T, and T/T was 1.7 times ungrafted. T/P also had a small late ROS burst under mock, earlier and stronger MAPK phosphorylation, and the highest resistance to Pst DC3000 among ungrafted, T/T and T/P plants. · Incompatible scions demonstrate increased PTI; Figure legends

3. 03

   ShownLowering SA does not rescue incompatibility: nahG tomato grafted with pepper still failed. nahG self-grafts healed poorly despite low SA, with high cell death and persistent defense gene activation, which places SA downstream of compatibility determination.

   With n = 10 grafts at 7 DAG, nahG self-grafts had almost no fully reconnected grafts and cell death similar to T-P heterografts. No disease signs appeared by 14 DAG, but adventitious roots formed. RNA-seq found 726 DEGs unique to the n/n scion and 477 unique to the n/n stock. SlPR1 was significantly higher than in T/T in both nahG self-graft tissues and in pepper-on-nahG stocks. · Loss of SA accumulation cannot rescue…; Figure legends

4. 04

   ShownHigh-dose exogenous SA on compatible tomato self-grafts phenocopies incompatibility. It blocks vascular reconnection, raises cell death and lowers junction auxin, repressing auxin biosynthesis, signaling and transport genes.

   Of 0.02, 0.05 and 0.1 mM SA, only 0.1 mM disrupted healing: 16.7% of grafts failed to adhere and 50% showed nonvascular adhesion only, with more trypan blue cell death. Auxin and YUC3, IAA9, SAUR32 and PIN1 expression fell as SA rose. WGCNA module ME1 (P = 9.6e-05), up only in compatible stocks, was topped by response to auxin. · Increased SA represses graft healing; SA inhibits auxin accumulation in the graft…; Figure legends

5. 05

   SuggestedIncompatible grafts accumulate less auxin at the junction. Exogenous auxin (IAA) only partly restores vascular differentiation and does not activate cambial regulators, so auxin alone cannot restore compatibility.

   pDR5:GUS signal appeared at 2 DAG in tomato self-grafts, only weakly at 4 DAG in tomato/pepper, and was absent from pepper/tomato reporter stocks. Auxin was significantly lower in T/P and P/T stocks. With 100 μM IAA, heterografts formed new but non-cohesive vascular tissue, auxin genes changed non-significantly, and cambial regulators (WOX4, PXY) did not rise. · Incompatible grafts have reduced auxin…; Exogenous auxin has limited, but significant…; Immune activation suppressed the necessary auxin…; Figure legends