Plant nucleotide binding/leucine-rich repeat (NLR) immune receptors are activated by pathogen effectors to trigger host defenses and cell death. Toll-interleukin 1 receptor domain NLRs (TNLs) converge on the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) family of lipase-like proteins for all resistance outputs. In Arabidopsis (Arabidopsis thaliana) TNL-mediated immunity, AtEDS1 heterodimers with PHYTOALEXIN DEFICIENT4 (AtPAD4) transcriptionally induced basal defenses. AtEDS1 uses the same surface to interact with PAD4-related SENESCENCE-ASSOCIATED GENE101 (AtSAG101), but the role of AtEDS1-AtSAG101 heterodimers remains unclear. We show that AtEDS1-AtSAG101 functions together with N REQUIRED GENE1 (AtNRG1) coiled-coil domain helper NLRs as a coevolved TNL cell death-signaling module. AtEDS1-AtSAG101-AtNRG1 cell death activity is transferable to the Solanaceous species Nicotiana benthamiana and cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and heterodimer structure-guided phenotyping of AtEDS1 variants and AtPAD4-AtSAG101 chimeras identify closely aligned ɑ-helical coil surfaces in the AtEDS1-AtSAG101 partner C-terminal domains that are necessary for reconstituted TNL cell death signaling. Our data suggest that TNL-triggered cell death and pathogen growth restriction are determined by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these signaling machineries coevolved with other components within plant species or clades to regulate downstream pathways in TNL immunity.

~200 words) 28 Plant intracellular nucleotide-binding/leucine-rich repeat (NLR) immune receptors are 29 activated by pathogen effectors to trigger host defenses and cell death. Toll-30 Interleukin1-receptor (TIR)-domain NLRs (TNLs) converge on the Enhanced Disease 31 Susceptibility1 (EDS1) family of lipase-like proteins for all resistance outputs. In 32 Arabidopsis TNL immunity, AtEDS1 heterodimers with Phytoalexin Deficient4 33 (AtPAD4) transcriptionally boost basal defense pathways. AtEDS1 uses the same 34 surface to interact with PAD4-related Senescence-Associated Gene101 (AtSAG101), 35 but the role of AtEDS1-AtSAG101 heterodimers was unclear. We show that AtEDS1-36 AtSAG101 function together with AtNRG1 coiled-coil domain helper NLRs as a 37 coevolved TNL cell death signaling module. AtEDS1-AtSAG101-AtNRG1 cell death 38 activity is transferable to the solanaceous species, Nicotiana benthamiana, and 39 cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with 40 endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and 41 heterodimer structure-guided phenotyping of AtEDS1 variants or AtPAD4-AtSAG101 42 chimeras identify closely aligned ɑ-helical coil surfaces in the AtEDS1-AtSAG101 43 partner C-terminal domains that are necessary for TNL cell death signaling. Our data 44 suggest that TNL-triggered cell death and pathogen growth restriction are determined 45 by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these 46 signaling machineries coevolved with further components within plant species or 47 clades to regulate downstream pathways in TNL immunity. 48 49

Summary Plant root architecture plasticity in response to biotic stresses has not been thoroughly investigated. Infection by endoparasitic cyst nematodes induces root architectural changes that involve the formation of secondary roots at infection sites. However, the molecular mechanisms regulating secondary root formation in response to cyst nematode infection remain largely unknown. We first assessed whether secondary roots form in a nematode density‐dependent manner by challenging wild‐type Arabidopsis plants with increasing numbers of cyst nematodes (Heterodera schachtii). Next, using jasmonate‐related reporter lines and knockout mutants, we tested whether tissue damage by nematodes triggers jasmonate‐dependent secondary root formation. Finally, we verified whether damage‐induced secondary root formation depends on local auxin biosynthesis at nematode infection sites. Intracellular host invasion by H. schachtii triggers a transient local increase in jasmonates, which activates the expression of ERF109 in a COI1‐dependent manner. Knockout mutations in COI1 and ERF109 disrupt the nematode density‐dependent increase in secondary roots observed in wild‐type plants. Furthermore, ERF109 regulates secondary root formation upon H. schachtii infection via local auxin biosynthesis. Host invasion by H. schachtii triggers secondary root formation via the damage‐induced jasmonate‐dependent ERF109 pathway. This points at a novel mechanism underlying plant root plasticity in response to biotic stress.

SUMMARY Infections by root‐feeding nematodes have profound effects on root system architecture and consequently shoot growth of host plants. Plants harbor intraspecific variation in their growth responses to belowground biotic stresses by nematodes, but the underlying mechanisms are not well understood. Here, we show that the transcription factor TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR‐9 (TCP9) modulates root system architectural plasticity in Arabidopsis thaliana in response to infections by the endoparasitic cyst nematode Heterodera schachtii. Young seedlings of tcp9 knock‐out mutants display a significantly weaker primary root growth inhibition response to cyst nematodes than wild‐type Arabidopsis. In older plants, tcp9 reduces the impact of nematode infections on the emergence and growth of secondary roots. Importantly, the altered growth responses by tcp9 are most likely not caused by less biotic stress on the root system, because TCP9 does not affect the number of infections, nematode development, and size of the nematode‐induced feeding structures. RNA‐sequencing of nematode‐infected roots of the tcp9 mutants revealed differential regulation of enzymes involved in reactive oxygen species (ROS) homeostasis and responses to oxidative stress. We also found that root and shoot growth of tcp9 mutants is less sensitive to exogenous hydrogen peroxide and that ROS accumulation in nematode infection sites in these mutants is reduced. Altogether, these observations demonstrate that TCP9 modulates the root system architectural plasticity to nematode infections via ROS‐mediated processes. Our study further points at a novel regulatory mechanism contributing to the tolerance of plants to root‐feeding nematodes by mitigating the impact of belowground biotic stresses.

Phytohormones play an essential role in different stages of plant-nematode interactions. Strigolactones (SLs) are a novel class of plant hormones which play an important role in plant development. Furthermore, certain soil-inhabiting organisms exploit this plant molecule as allelochemical. However, whether SLs play a role in plant parasitism by nematodes is as yet unknown. This prompted us to investigate the potential role of SLs in different stages of the nematode life cycle using the beet cyst nematode Heterodera schachtii and Arabidopsis as a model system. We analyzed the effect of SLs on cyst nematode hatching, host attraction and invasion, and the establishment of a feeding relation upon infection of the SL deficient mutant max4-1 and the SL signaling mutant max2-1. In addition, infection assays were performed under phosphate shortage to enhance SL production and in the presence of the synthetic SL analog GR24. From this study, we can conclude that SLs do not contribute to cyst nematode hatching at the levels tested but that they do play a role in host attraction and subsequent invasion in a MAX2 dependent manner. Furthermore, we observed that increased levels of exogenous and endogenous SLs change the root invasion zone. Upon root infection, cyst nematode development was enhanced in both the max2-1 and max4-1 mutants due to the formation of enlarged feeding cells. These data provide evidence for distinct roles of SLs during cyst nematode parasitism of plant roots.