SIX1 (Secreted in xylem 1), also called Avr3, is a secreted effector of the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. The gene encodes a 284-residue (about 32 kDa) precursor with an N-terminal signal peptide and a pro-domain followed by a cysteine-rich mature region; a 12 kDa cysteine-rich species derived from the central part of the protein was the first fungal protein identified in the xylem sap of infected tomato. The gene is expressed almost exclusively during host infection, is strongly induced on contact with tomato roots and in hyphae colonising xylem vessels, and depends on the nuclear regulator Sge1. Six1 is required for full virulence on susceptible tomato, but its host target and biochemical mechanism are unknown. In tomato carrying the I-3 resistance gene, which encodes a plasma-membrane S-receptor-like kinase, Six1 acts as the avirulence determinant: SIX1 deletion breaks I-3-mediated resistance and reintroduction restores it. Complementation with homologues from a cape-gooseberry-infecting forma specialis showed that recognition by I-3 can be separated from the virulence activity, which suggests direct recognition. I-3-mediated recognition of Six1 is suppressed by another effector, Avr1 (SIX4). The crystal structure of Six1 (residues 22-284) shows a disulfide-stabilised two-domain fold, an N-terminal domain of one alpha-helix and five beta-strands plus a C-terminal beta-sandwich, which defines (with Avr1) the FOLD (Fol dual-domain) effector family. No enzymatic or other molecular activity has been demonstrated.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0020002 host cell plasma membrane | IDA PMID:25740416 The tomato I-3 gene: a novel gene for resistance to Fusarium... | UNDECIDED | Summary: PHI-base IDA annotation from the paper that cloned tomato I-3 and showed that it encodes an S-receptor-like kinase (SRLK) conferring Avr3-dependent resistance. Only the abstract is cached and no PMC full text exists. The abstract reports no localisation experiment on Six1/Avr3; the plasma-membrane protein in this system is the receptor I-3, and Six1 itself has so far been detected only in xylem sap, outside host cells. Reason: An IDA localisation of Six1 to the host plasma membrane would be a notable finding, since Six1 is known as a xylem-sap (apoplastic) protein and the I-3 receptor is the plasma-membrane component. The cached abstract describes only map-based cloning and transgenic complementation, and does not state that Six1 was localised. It is possible that the annotation reflects localisation of I-3 rather than Six1, or a Six1-binding or localisation experiment in the full text. I could not read the full text, so per policy this is left UNDECIDED rather than removed. Supporting Evidence: PMID:25740416 Here, we describe the fine mapping and sequencing of genes at the I-3 locus, which revealed a family of S-receptor-like kinase (SRLK) genes. PMID:15387816 The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. |
| GO:0043657 host cell | EXP PMID:23596453 A nuclear localization for Avr2 from Fusarium oxysporum is r... | REMOVE | Summary: PHI-base annotation from Ma et al., a full-text paper about the nuclear localisation of a different effector, Avr2 (SIX3). In this paper SIX1/AVR3 appears only as a promoter-reporter: the fungal strain used has the Avr3 coding sequence replaced by GFP, so GFP marks AVR3 promoter activity in fungal hyphae penetrating roots and growing in xylem vessels. No Six1 protein was localised, and nothing places Six1 inside host cells. Reason: Unlike the abstract-only case above, the full text is cached and every mention of SIX1/AVR3 was checked. The only SIX1-related experiment is pAVR3:GFP reporter expression, which reports fungal gene expression in fungal hyphae, not the location of the Six1 protein (which is not even produced in that strain). Biologically, Six1 is a xylem-sap effector detected outside host cells and perceived by a cell-surface receptor, and no study has shown uptake into host cells. "host cell" therefore has no support from this reference or elsewhere. The host-cell localisation in this paper belongs to Avr2. The extracellular location is captured by the NEW host apoplast annotation below. Supporting Evidence: PMID:23596453 In this strain the coding sequence for Avr3 has been replaced by GFP encoding Green Fluorescent Protein (Van Der Does et al., 2008). The advantage of employing the Fol-pAVR3:GFP strain is that GFP can be used to monitor the growth of Fol in roots PMID:23596453 From three days after inoculation and onward high expression of both AVR3 and AVR2 was found in hyphae growing in the xylem vessels of tomato roots. file:FUSOX/SIX1/SIX1-deep-research-falcon.md No convincing evidence places Fol Six1 inside tomato cells. |
| GO:0140404 effector-mediated perturbation of host innate immune response by symbiont | EXP PMID:15672814 Fusarium oxysporum evades I-3-mediated resistance without al... | ACCEPT | Summary: Rep et al. (2005) showed that the SIX1 gene of an I-3-virulent isolate is fully functional: its product is secreted in xylem sap, its deletion further increases virulence on the I-3 line and reduces virulence on susceptible lines, and it confers full avirulence on the I-3 line when moved into another genetic background. Reason: Gene deletion and transfer experiments on SIX1 itself show that the secreted protein triggers I-3-dependent immunity (avirulence) and also contributes to virulence on susceptible plants. GO:0140404 is defined as modulation, either activation or suppression, of a host innate immune response by a secreted symbiont molecule. This neutral parent is the right level for Six1. Its recognition activates I-3 immunity, but HR-type cell death has not been shown, so GO:0080185 (activation of plant hypersensitive response) would overstate the evidence. No immunosuppressive activity of Six1 has been shown, so a suppression term would also overstate it. Supporting Evidence: PMID:15672814 deletion leads to a further increase in virulence on the I-3 line as well as reduced virulence on susceptible lines, and iii) the gene confers full avirulence on the I-3 line when transferred to another genetic background. PMID:15387816 Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. |
| GO:0140404 effector-mediated perturbation of host innate immune response by symbiont | EXP PMID:18464895 Suppression of plant resistance gene-based immunity by a fun... | ACCEPT | Summary: Houterman et al. (2008) identified Avr1 (SIX4) as a suppressor of I-2- and I-3-mediated resistance. They used an AVR3 (SIX1) gene-replacement strain (Fol004 avr3 deletion) to show that I-3 recognition depends on Avr3, and noted that Avr3 is required for full virulence on susceptible plants. Reason: The paper includes experiments with a SIX1 deletion strain that confirm Avr3-dependent I-3 resistance and the contribution of Avr3 to virulence. This supports the neutral perturbation term, as for the 2005 paper above. The immunosuppressive activity described in this paper belongs to Avr1, not to Six1, and should not be transferred to Six1. Supporting Evidence: PMID:18464895 In (F), Fol004 avr3Ξ (race 1 strain Fol004 with AVR3 (SIX1) deleted by gene replacement) was used. PMID:18464895 During evolution of the tomato-Fol pathosystem, I-2 and I-3 have evolved to recognize, respectively, Avr2 and Avr3. Since Avr3 is required for full virulence of Fol, evasion of I-3 recognition through loss of the AVR3 gene would entail a serious fitness penalty. |
| GO:0140404 effector-mediated perturbation of host innate immune response by symbiont | EXP PMID:25740416 The tomato I-3 gene: a novel gene for resistance to Fusarium... | ACCEPT | Summary: Catanzariti et al. (2015) cloned I-3 and showed by transgenic complementation that one SRLK gene confers Avr3-dependent resistance to Fol race 3. Reason: Avr3-dependent resistance conferred by the cloned I-3 gene shows from the host side that Six1 activates I-3 immunity. Abstract-only access, but the abstract states the Avr3 dependence explicitly, and the term (neutral perturbation) matches the documented role. Supporting Evidence: PMID:25740416 Transgenic tomato lines were generated with three of these SRLK genes and one was found to confer Avr3-dependent resistance to Fol race 3, confirming it to be I-3. |
| GO:0140404 effector-mediated perturbation of host innate immune response by symbiont | EXP PMID:26177154 Identification of I-7 expands the repertoire of genes for re... | REMOVE | Summary: Gonzalez-Cendales et al. (2016) identified I-7, an LRR-RLP gene for resistance to Fol race 3. The cached full text has no experiment on SIX1: Avr3 is mentioned only in the list of strains (race 3 isolate "carrying Avr3") and in discussion of how Avr1 suppresses I-2 and I-3. The effector recognised by I-7 (Avr7) is a different, then unidentified protein. Reason: The term itself is correct for Six1 and is kept through the three rows above. This row is removed because the cited paper has no Six1 experiment; full text was checked. Six1 was not deleted, expressed or assayed, and I-7 resistance is triggered by a different effector. The paper's statement that Avr1 does not suppress I-7 says nothing about Six1 activity. Supporting Evidence: PMID:26177154 Fol race 3 isolate #1943 (carrying Avr3 ) was provided by Mr Des McGrath (AgriβScience Queensland, Australia) PMID:26177154 It is possible that the action of Avr1 is limited to Avr2 and Avr3 through a functional relationship between the three effectors |
| GO:0140593 host apoplast | IDA PMID:15387816 A small, cysteine-rich protein secreted by Fusarium oxysporu... | NEW | Summary: Six1 was identified by mass spectrometry as a 12 kDa cysteine-rich protein in the xylem sap of infected tomato. Xylem vessel lumens are part of the plant apoplast, and Six1 is perceived there by the cell-surface receptor I-3. Reason: Location annotation, so the process participation test does not apply. GOA has no supported location for Six1 (the host cell row is removed and the host plasma membrane row is unverified), although secretion into xylem sap is how the protein was discovered and named. The GO:0140593 definition explicitly includes the lumen of xylem vessels. The same term is used for other apoplastic fungal effectors, for example Fulvia fulva AVR9 in this repository. Supporting Evidence: PMID:15387816 A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. PMID:15387816 The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. PMID:15672814 the gene product is secreted in xylem sap |
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Download this section (compressed HTML)Q: Does the host cell plasma membrane IDA (PHI-base, PMID:25740416) reflect an experiment localising Six1 itself, for example binding to I-3-expressing cells, or the localisation of the I-3 receptor? Six1 has otherwise been found only in xylem sap.
Q: GO has no pathogen-side term for "activation of host immune response by recognition of the effector" without hypersensitive cell death, and GO:0080185 sits under a suppression parent. Is the neutral GO:0140404 the intended term for avirulence effectors whose recognition activates immunity without HR, such as Six1/I-3?
Q: Which host process does Six1 act on to promote virulence on susceptible tomato, and is this distinct from the surface that I-3 recognises (the hypervariable V130-T184 region)?
Experiment: Test direct binding of purified mature Six1 (and the non-recognised homologue SIX1a as a control) to the I-3 S-receptor ectodomain, for example by co-immunoprecipitation from Nicotiana benthamiana or by in vitro binding assays, to establish whether recognition is direct.
Experiment: Identify Six1 host interactors in tomato xylem sap and root apoplast by affinity purification-mass spectrometry, and test candidate targets for involvement in the virulence role using SIX1 deletion and complementation strains.
Experiment: Swap the hypervariable region between Fol SIX1 and Foph SIX1a/SIX1b and test the chimeras for I-3 avirulence and for restoration of virulence on susceptible tomato, to map the recognition and virulence determinants.
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