Inhibition of jasmonate-mediated plant defences by the fungal metabolite higginsianin B.

Infection of Arabidopsis thaliana by the ascomycete fungus Colletotrichum higginsianum is characterised by an early symptomless biotrophic phase followed by a destructive necrotrophic phase. The fungal genome contains 77 secondary metabolism-related biosynthetic gene clusters (BGCs), and their expression during the infection process is tightly regulated. Deleting CclA, a chromatin regulator involved in repression of some BGCs through H3K4 trimethylation, allowed overproduction of 3 families of terpenoids and isolation of 12 different molecules. These natural products were tested in combination with methyl jasmonate (MeJA), an elicitor of jasmonate responses, for their capacity to alter defence gene induction in Arabidopsis. Higginsianin B inhibited MeJA-triggered expression of the defence reporter VSP1p:GUS, suggesting it may block bioactive JA-Ile synthesis or signalling in planta. Using the JA-Ile sensor Jas9-VENUS, we found that higginsianin B, but not three other structurally-related molecules, suppressed JA-Ile signalling by preventing degradation of JAZ proteins, the repressors of JA responses. Higginsianin B likely blocks the 26S proteasome-dependent degradation of JAZ proteins because it inhibited chymotrypsin- and caspase-like protease activities. The inhibition of target degradation by higginsianin B also extended to auxin signalling, as higginsianin B treatment reduced IAA-dependent expression of DR5p:GUS. Overall, our data indicate that specific fungal secondary metabolites can act similarly to protein effectors to subvert plant immune and developmental responses.


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The perception of microbial plant aggressors is mediated by the recognition of pathogen-associated 45 molecular patterns (PAMPs) by plant cell surface receptors, which in turn activates a cascade of 46 PAMP-triggered immune (PTI) responses (Dodds and Rathjen 2010;Zipfel and Robatzek 2010). 47 Downstream of PTI activation, these immune responses are regulated by an interconnected network of 48 phytohormone signalling pathways in which jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) 49 play a central role (Pieterse et al., 2012). Antagonistic and synergistic interactions between these 50 pathways provide an additional layer of regulation in which hormone cross-talk allows the plant to 51 fine-tune its immune responses to particular pathogens (Bigeard et al., 2015, Pieterse et al., 2012. A 52 broad range of microbes target these hormones signalling pathways using secreted protein or small 53 molecule effectors in order to manipulate or circumvent plant immunity (Plett et al. 2014;Patkar et 54 al., 2015;Gimenez-Ibanez et al. 2016;Katsir et al. 2008;Groll et al. 2008;Stringlis et al., 2018). 55 The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease in numerous wild 56 and cultivated members of the Brassicaceae, including Arabidopsis thaliana. The latter interaction 57 provides a model pathosystem in which both partners are amenable to genetic manipulation and rich 58 genetic resources are available for the plant host. Infection of A. thaliana by C. higginsianum is 59 characterised by an early symptomless biotrophic phase followed by a destructive necrotrophic phase 60 (O'Connell et al., 2004). As with other hemibiotrophic pathogens, it is assumed that during the has allowed the isolation of numerous novel metabolites from diverse axenically grown fungi (e.g.

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Despite the huge efforts made in recent years to characterise the natural products produced by plant-  (Meesters et al., 2014, Serrano et al., 2015. Using a forward chemical genetic screen, 83 we here identify a fungal natural product that suppresses JA-mediated plant defences. Using different 84 JA-reporter lines in Arabidopsis, we show that higginsianin B, a terpenoid metabolite produced by C. 85 higginsianum, can prevent the MeJA-dependent degradation of JAZ repressor proteins. Three 86 structural analogues of higginsianin B were found to lack this activity, providing clues to the structure-87 activity relationship and suggesting candidate functional groups which could help in identifying target 88 binding sites. We also found that the active metabolite is able to inhibit the plant developmental 89 signalling pathway mediated by auxin. Finally, we present evidence that higginsianin B is likely to 90 exert its activity through inhibition of the 26S proteasome. Taken together, our work highlights the 91 importance of fungal secondary metabolites in manipulating plant hormone signalling. The Colletotrichum higginsianum wild-type (WT) strain (IMI 349063A) was maintained on Mathur's 96 medium as previously described (O'Connell et al., 2004). Arabidopsis thaliana accession Columbia 97 (Col-0) was used as the WT line and served as genetic background for the previously described   MeJA treatment. In this way, expression of the reporter was evaluated in individual seedling roots (n 135 = 10 for each condition). To ensure that pre-treatments did not cause reporter degradation, a full sample 136 set was also pre-treated directly on microscope slides and imaged at 0 min and after 30 min. VENUS 137 fluorescence in living roots was imaged with a Zeiss LSM 700 confocal laser scanning microscope 138 with 488 nm excitation and 490-555 nm emission wavelength. All images shown within one 139 experiment were taken with identical settings. Image processing was done with FIJI (http://fiji.sc/Fiji). Five-day-old seedlings were grown horizontally in axenic conditions on a nylon mesh (200 µm pore 144 size) supported on MS solid medium. Growth conditions were 21°C with a photoperiod of 14h light 145 (100 µE•m -2 •s -1 ). Pre-treatment and treatment of seedlings was performed as described for microscopy, 146 except that treatments were performed in sterile dishes. E-64, a highly selective cysteine protease 147 inhibitor (E3132, Sigma-Aldrich) and epoxomicin, a specific proteasome inhibitor (E3652, Sigma-148 Aldrich) were used as controls. Seedlings were snap-frozen in liquid nitrogen and kept frozen for   Conover-Iman test with Benjamini-Hochberg adjustment of P-values for false discovery rate (FDR).

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All proteasome activity tests were performed at least in duplicate and data were statistically analysed 195 with an ANOVA single factor test.  Figure S2). Control seedlings that were not treated with MeJA (uninduced) displayed 221 only basal activation of VSP1p:GUS (8% of the level in induced seedlings, Figure 1A). Using this 222 assay, we also found that higginsianin B reduced VSP1p:GUS activation in a dose-dependent manner 223 between 3 and 100 µM, with maximal inhibition of 56% at 100 µM ( Figure 1B). Given the pronounced 224 inhibitory effect of higginsianin B on the JA pathway, we investigated this activity further. 226 To validate the primary screen result, we tested the effect of higginsianin B on a different marker of poly-ubiquitinated prior to degradation by the 26S proteasome (Chini et al., 2007, Thines et al., 2007. 234 We therefore monitored JAZ1-GUS protein degradation in roots pre-treated with test compounds and  Figure S3). A dose-dependency test showed that 268 higginsianin B was active at a concentration of 10 µM ( Figure 3D). As controls in this assay, E-64, a    289 The degradation of JAZ proteins is executed by the 26S proteasome upon poly-ubiquitination by 290 SCF COI1 complex (Chini et al., 2007, Thines et al., 2007. Likewise, the 26S proteasome is also 291 involved in auxin perception by co-receptors, the SCF TIR1/AFB ubiquitin ligases and their targets, the 292 AUX/IAA family of auxin response inhibitors (Gray et al., 2001, Tiwari et al., 2001. If higginsianin 293 B blocks JAZ degradation by inhibiting proteasome activity, we reasoned that it may also impact other  Figure S4B). This 300 finding supports the hypothesis that higginsianin B could affect other proteasome-dependent 301 processes, such as the activation of auxin signalling.

The 26S proteasome is a target of higginsianin B 303
The impact of higginsianin B on JA-and auxin-mediated signalling pathways suggested the ubiquitin- caspase-like activities in a dose-dependent manner. The chymotrypsin-like activity was reduced to 316 ~60% at 24 h and ~50% at 48 h relative to the control ( Figure 5C). Caspase-like activity was strongly 317 reduced to 35% of the control at 24 h, but only to 70% of the control at 48 h ( Figure 5D). Overall, 318 these results suggest that higginsianin B is a potent inhibitor of proteasome proteolytic activities.

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To date, few chemical genetic screens have been used to systematically search for molecules 321 interfering with components of plant immunity (Dejonghe and Russinova 2017, Serrano et al., 2015.

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The first small molecule found to inhibit JA-mediated responses in a chemical screen was Jarin-1, a 323 plant-derived alkaloid that was subsequently shown to specifically inhibit the activity of JA-Ile 324 synthetase JAR1, thereby blocking the conversion of JA into bioactive JA-Ile (Meesters et al., 2014).

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Adopting a similar approach combined with bioassay-guided purification to screen secondary

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To gain insight into the structural features of higginsianin B that are required for its activity, we tested 339 the three other known members of this compound family, namely higginsianin A, C and 13-epi-340 higginsianin C. Higginsianin B has a bicyclic core substituted by hydroxyl and 4-isoheptenyl groups.

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In contrast, the three other molecules have a tricyclic core structure with the third ring being a 342 tetrahydrofuran substituted by either an isobutenyl group in the case of higginsianin A or an 343 isopropanol group in the case of higginsianin C and 13-epi-higginsianin C. Remarkably, higginsianin 344 B was the only molecule to show activity in JAZ degradation assays suggesting that either the hydroxyl 345 or the 4-isoheptenyl substituents of the bicyclic core (or both) contribute to the activity of higginsianin 346 B. On the other hand, a second hydroxyl group located on the pyrone ring in all higginsianins is 347 unlikely to contribute to this activity, and is therefore a good candidate for tagging higginsianin B with 348 a fluorescent probe for direct visualization of the active metabolite by live-cell imaging. This group 349 could also be exploited for the covalent immobilization of higginsianin B onto a solid support to search 350 for potential protein targets by affinity purification.

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In conclusion, our findings raise the possibility that higginsianin B could function during infection as 386 a chemical effector to suppress JA-mediated defences, which are induced at the necrotrophic phase of 387 C. higginsianum infection on Brassica and Arabidopsis (Narusaka et al. 2004;Narusaka et al. 2006).

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Work is now ongoing to determine at what stage higginsianin B is produced during infection and to 389 genetically test its contribution to fungal virulence and plant defence suppression. The authors are sincerely grateful to Erich Kombrink for valuable discussions. This work was 400 supported by "Chaire d'Excellence" FUNAPP grant (ANR-12-CHEX-0008-01) from the Agence  B pre-treatment is dose-dependent. Bars represent means VSP1p:GUS activity of 12 independent seedlings, 538  SD from one representative experiment performed twice. RLU: Relative Light Unit. **: adjusted P-value 539 < 0.01; ***: adjusted P-value < 0.001 (Kruskal-Wallis with Conover-Iman test). 540 Ile (lower row). The proteasome inhibitor MG132 was used as a known inhibitor of JAZ1-GUS degradation. 549 Each treatment was performed on at least 5 seedlings and one representative image is presented for each 550 treatment. 551 following 30min pre-treatments with mock or higginsianin B (higB) combined with mechanical wounding. 574 Shoots and roots were collected independently 1 h after wounding aerial organs. JAZ10 transcript levels 575 were normalised to those of UBC21 and displayed relative to the expression of mock controls. Bars 576 represent the means of three biological replicates (±SD), each containing a pool of organs from ∼60 577 seedlings. ns, not significant (P-value = 0.08, t-test); *: P-value < 0.05 (t-test). like (panel C) and caspase-like (panel D) proteasomal activities in BJ cells exposed for 24 h and 48 h to 585 higginsianin B. Data points correspond to the mean of the independent experiments and error bars denote 586 standard deviation (SD). FLU, Fluorescence unit. *: P-value < 0.05; **: P-value < 0.01 (ANOVA test). 587