Plants are master regulators of rhizosphere ecology, secreting a complex mixture of compounds into the soil, collectively termed plant root exudate. Root exudate composition is highly dynamic and functional, mediating economically important interactions between plants and a wide range of soil organisms. Currently we know very little about the molecular basis of root exudate composition, which is a key hurdle to functional exploitation of root exudates for crop improvement. Root expressed transporters modulate exudate composition and could be manipulated to develop beneficial plant root exudate traits. Using Virus Induced Gene silencing (VIGS), we demonstrate that knockdown of two root-expressed ABC transporter genes in tomato cv. Moneymaker, ABC-C6 and ABC-G33, alters the composition of semi-volatile compounds in collected root exudates. Root exudate chemotaxis assays demonstrate that knockdown of each transporter gene triggers the repulsion of economically relevant Meloidogyne and Globodera spp. plant parasitic nematodes, which are attracted to control treatment root exudates. Knockdown of ABC-C6 inhibits egg hatching of Meloidogyne and Globodera spp., relative to controls. Knockdown of ABC-G33 has no impact on egg hatching of Meloidogyne spp. but has a substantial inhibitory impact on egg hatching of G. pallida. ABC-C6 knockdown has no impact on the attraction of the plant pathogen Agrobacterium tumefaciens, or the plant growth promoting Bacillus subtilis, relative to controls. Silencing ABC-G33 induces a statistically significant reduction in attraction of B. subtilis, with no impact on attraction of A. tumefaciens. By inoculating selected differentially exuded compounds into control root exudates, we demonstrate that hexadecaonic acid and pentadecane are biologically relevant parasite repellents. ABC-C6 represents a promising target for breeding or biotechnology intervention strategies as gene knockdown leads to the repulsion of economically important plant parasites and retains attraction of the beneficial rhizobacterium B. subtilis. This study exposes the link between ABC transporters, root exudate composition, and ex planta interactions with agriculturally and economically relevant rhizosphere organisms, paving the way for new approaches to rhizosphere engineering and crop protection.

19Plants are master regulators of rhizosphere ecology, secreting a complex mixture of compounds into 20 the soil, collectively termed plant root exudate. Root exudate composition is highly dynamic and 21 functional, mediating interactions between plants and a wide range of beneficial / harmful soil 22 organisms. Exudate composition is under selective pressure to diversify in response to pathogen 23 perception, whilst maintaining interactions with beneficial organisms. However, crop domestication 24 has exerted significant and unintended changes to crop root exudate composition, and we know very 25 little about genotype -phenotype linkages that pertain to root exudates and rhizosphere interactions. 26 Better understanding could enable the modulation of root exudate composition for crop improvement 27 by promoting positive, and impeding negative, interactions. Root expressed transporters modulate 28 exudate composition and could be manipulated towards the rational design of beneficial root exudate 29 profiles. Using Virus Induced Gene silencing (VIGS), we demonstrate that knockdown of two root-30 expressed ABC transporter genes in tomato cv. Moneymaker, ABC-G33 and ABC-C6, alters the 31 composition of semi-volatile compounds in collected root exudates. Root exudate chemotaxis assays 32 demonstrate that knockdown of each transporter gene triggers the repulsion of economically relevant 33 Meloidogyne and Globodera spp. plant parasitic nematodes, which are attracted to control treatment 34 root exudates. Knockdown of ABC-C6 inhibits egg hatching of Meloidogyne and Globodera spp., 35 relative to controls. Knockdown of ABC-G33 has no impact on egg hatching of Meloidogyne spp. but 36 has a substantial inhibitory impact on egg hatching of G. pallida. ABC-C6 knockdown has no impact 37 on the attraction of the plant pathogen Agrobacterium tumefaciens, or the plant growth promoting 38 Bacillus subtilis, relative to controls. Silencing ABC-G33 induces a statistically significant reduction in 39 attraction of B. subtilis, with no impact on attraction of A. tumefaciens. ABC-C6 represents a promising 40 target for breeding or biotechnology intervention strategies as gene knockdown (-64.9%) leads to the 41 repulsion of economically important plant parasites and retains attraction of the beneficial 42 rhizobacterium B. subtilis. This study exposes the link between ABC transporters, root exudate 43 composition, and ex planta interactions with agriculturally and economically relevant rhizosphere 44 organisms, paving the way for an entirely new approach to rhizosphere engineering and crop 45 protection. 46 47 560 VIGS plasmids pTRV1 and pTRV2 were kindly provided by Prof. Ian Graham, University of York. A.

Plant root exudates are compositionally diverse, plastic and adaptive. Ethylene signalling influences the attraction of plant parasitic nematodes (PPNs), presumably through the modulation of root exudate composition. Understanding this pathway could lead to new sources of crop parasite resistance. Here we used Virus-Induced Gene Silencing (VIGS) to knock down the expression of two Ethylene Response Factor (ERF) genes, ERF-E2 and ERF-E3, in tomato. Root exudates were significantly more attractive to the PPNs Meloidogyne incognita and Globodera pallida following knockdown of ERF-E2, which had no impact on the attraction of Meloidogyne javanica. Knockdown of ERF-E3 had no impact on the attraction of Meloidogyne or Globodera spp. Gas Chromatography Mass Spectrometry (GC-MS) analysis revealed major changes in root exudate composition relative to controls. However, these changes did not alter the attraction of rhizosphere microbes Bacillus subtilis or Agrobacterium tumefaciens. This study further supports the potential of engineering plant root exudate for parasite control, through the modulation of plant genes.

Meloidogyne incognita is an economically important plant parasitic nematode. Here we demonstrate substantial variation in the invasiveness of four M. incognita populations relative to tomato. Infective (J2) stage transcriptomes reveal significant variation in the expression of proteincoding and non-coding RNAs between populations. We identify 33 gene expression markers (GEMs) that correlate with invasiveness, and which map to genes with predicted roles in host-finding and invasion, including neuropeptides, ion channels, GPCRs, cell wall-degrading enzymes and microRNAs. These data demonstrate a surprising diversity in microRNA complements between populations, and identify GEMs for invasiveness of M. incognita for the first time.

11Meloidogyne incognita is an economically important plant parasitic nematode. Here we 12 demonstrate substantial variation in the invasiveness of four M. incognita populations relative to 13 tomato. Infective (J2) stage transcriptomes reveal significant variation in the expression of protein-14 coding and non-coding RNAs between populations. We identify 33 gene expression markers (GEMs) 15 that correlate with invasiveness, and which map to genes with predicted roles in host-finding and 16 invasion, including neuropeptides, ion channels, GPCRs, cell wall-degrading enzymes and microRNAs. 17These data demonstrate a surprising diversity in microRNA complements between populations, and 18 identify GEMs for invasiveness of M. incognita for the first time. 19 20