NaMLP, a new identified Kunitz trypsin inhibitor regulated synergistically by JA and ethylene, confers Spodoptera litura resistance in Nicotiana attenuata

doi:10.21203/rs.3.rs-2130754/v1

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NaMLP, a new identified Kunitz trypsin inhibitor regulated synergistically by JA and ethylene, confers Spodoptera litura resistance in Nicotiana attenuata

https://doi.org/10.21203/rs.3.rs-2130754/v1

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published 06 Feb, 2023

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Upon insect herbivore attack, Nicotiana attenuata accumulates trypsin protease inhibitor (TPI) activities as a defense response from different protease inhibitor (PI) coding genes, including WRKY3-regulated NaKTI2, and JA-dependent NaPI. However, whether any other TPI gene exists in N. attenuata is still unclear. A miraculin-like protein gene (NaMLP) was highly up-regulated in N. attenuata after Alternaria alternata infection. However, silencing NaMLP had no effect on lesion diameter developed on N. attenuata leaves after A. alternata inoculation. Meanwhile, the transcripts of NaMLP could be induced by wounding and amplified by Spodoptera litura oral secretions (OS). S. litura larvae gained significantly more biomass on NaMLP-silenced plants but less on NaMLP over-expressed plants. Although NaMLP showed low sequence similarity to NaKTI2, it had conserved reaction sites of Kunitz trypsin inhibitors, and exhibited TPI activities when its coding gene was over-expressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP over-expressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficent irAOC and ethylene-reduced irACO plants, and the expression of NaMLP could be significantly induced by methyl jasmonate or ethephon alone, but dramatically amplified by co-treatment of both methyl jasmonate and ethephon. Thus, our results demonstrate that in addition to JA-regulated NaPI, and WRKY3/6-dependent NaKTI2, N. attenuata plants also up-regulates TPI activities via NaMLP, which confers S. litura resistance through JA and ethylene signaling in a synergistic way.

Ethylene

jasmonate

miraculin-like protein

Nicotiana

Spodoptera

trypsin protease inhibitor

Plant protease inhibitors (PIs) have been well established to play an important defensive role against insect herbivores and pathogens. They include Kunitz, Bowman-Birk, Potato I and II, Squash, Barley, Cystatins and Miscellaneous type. In wild tobacco Nicotiana attenuata, NaPI, which belongs to the potato PI-II family and exhibits trypsin protease inhibitor (TPI) activities, was identified as an effective defense against plant’s native herbivores Manduca sexta (Zavala, et al. 2004).

Most Kunitz-type trypsin inhibitors (KTIs) have four conserved cysteine residues that form two disulfide bonds, and have been reported to localize in cell wall of elongating seedling organs and vascular tissue (Jimenez, et al. 2007). KTIs are generally strongly induced by injury or insects feeding (Eberl, et al. 2021). Spodoptera litura is a serious polyphagous pest, infesting various host plants of economic importance (Bhattacharyya, et al. 2007). Recently, a KTI (NaKTI2) is found to be specifically elicited in N. attenuata by oral secretions (OS) of S. litura and confers S. litura resistance (Yin, et al. 2021), indicating N. attenuata plants accumulate different PIs, including NaPI and NaKTI2 to defense against insect herbivores.

Plant hormones play key regulatory roles in plant defense responses, especially JA and ET signaling (Chen, et al. 2021). JA is an essential signal positively regulating defense responses of N. attenuata to insect herbivores. TPI activities were dramatically reduced in JA-insensitive irCOI1 plants (Paschold, et al. 2007). Insect herbivore-induced NaPI was completely dependent on JA signaling, since it was abolished in JA-deficient irAOC plants (Yin, et al. 2021). However, NaKTI2 was only slightly regulated by JA signaling, but mainly dependent on transcription factor NaWRKY3 and NaWRKY6 (Yin, et al. 2021).

Alternaria alternata is a notorious necrotrophic fungus causing black spot disease in Nicotiana species. When infected by A. alternata, N. attenuata plants will accumulate certain amounts of phytoalexin scopoletin and scopolin to defend against this pathogen (Li and Wu 2016; Sun, et al. 2014). Feruloyl-CoA 6ʹ-hydroxylase 1 (F6ʹH1) is the gene encoding the key enzyme for scopoletin biosynthesis. Both JA and ethylene signaling are required for A. alternata-elicited F6ʹH1 expression (Sun, et al. 2017; Sun, et al. 2014).

Miraculin is a taste-modifying protein, it has been known for a long time due to its surprising property of transforming sourness into sweetness (Mohammad, et al. 2011). Interestingly, many miraculin-like proteins (MLPs), which have certain sequence similarity to natural miraculin, have been reported to be involved in pest and pathogen resistance. RlemMLP2, but not RlemMLP1 could inhibit the growth of Alternaria spores (Tsukuda, et al. 2006). In curry tree, MKMLP can resist Helicoverpa armigera and S. litura by inhibiting the activity of intestinal protease (Gahloth, et al. 2011).

In this study, a miraculin-like protein (NaMLP) which is strongly induced in N. attenuata after A. alternata infection draws our attention. By generation of NaMLP-silenced and over-expressed lines, we found that NaMLP was required for the resistance against S. litura instead of A. alternata. Further sequence alignment and functional tests revealed that NaMLP is a new KTI. Finally we demonstrate that NaMLP is a KTI regulated synergistically by JA and ethylene in N. attenuata.

2.1 Plant and fungal material

An inbred line of N. attenuata was used as the wild-type (WT). Stably transformed lines of JA-deficient plant irAOC, JA-insensitive plant irCOI1, ethylene-reduced plant irACO, and ethylene-insensitive plant Ov-etr1 were provided by Professor Ian T. Baldwin (Max-Planck Institute of Chemical Ecology); Plants silenced with NaMLP (irNaMLP line 11 and 12), over-expressed with NaMLP (OvNaMLP line 2 and 3) were generated in this study. Seed germination and plant growth were conducted as described (Krügel, et al. 2002; Zhao, et al. 2021). A. alternata were grown and used for inoculation as described(Sun, et al. 2014; Sun, et al. 2014).

2.2 S. litura performance and leaf treatments

S. litura eggs were purchased from Keyun biological company and hatched in a growth chamber at 26 ℃ for 16-h light, and 24 ℃ for 8-h dark as described (Wu, et al. 2013). To investigate how S. litura larvae performed on plants, 5 newly hatched neonates were randomly selected and placed directly on node + 3 leaves (0, + 1, + 2 leaves were used for real-time PCR or TPI activity experiments) of plants; 24 plants for each genotype were used for caterpillar performance. The weight of S. litura larvae was recorded. The caterpillar performance experiments were repeated three times with similar tendency, and one of them was presented in this study.

To analyze gene expression and TPI activity, wounding plus water (W + W) or wounding plus oral secretions (W + OS) of S. litura larvae treatments were used to mimic herbivory. For simulated herbivory treatments, the source–sink transition leaves (0 leaves) were wounded with a fabric pattern wheel and 20 µl OS of S. litura larvae (diluted 3 times) or water were rapidly rubbed into the puncture wounds of each wounded leaf (Halitschke and Baldwin 2003).

2.3 Sequence analysis

Miraculin (Synsepalum dulcificum, BAH84844), NaKTI2 (N. attenuata, XP_019230715.1) and NtKTI (N. tabacum, ACL12055) were used for sequence alignment with NaMLP (N. attenuata, XP_019229306.1). Multiple protein alignment was performed by MEGA-Ⅵ and GeneDoc applications. Secondary and tertiary structure predictions were made by JPred and SWISSMODEL, which were used to manually edit and mark in the alignment according to (Yin, et al. 2021).

2.4 Transient over-expression and trypsin protease inhibitor (TPI) activity assay

The open reading frame of NaMLP was amplified by primers (CJ09 and CJ10) and insert directly upstream of eGFP under the control of CaMV 35S promoter in pCAMBIA1301. Agrobacterium tumefaciens (strain GV3101) cells carrying this construct or empty vector (EV) were inoculated into the leaves of 6-week-old Ov-NahG N. attenuata plants for transient expression. Inoculated leaves were collected after 48 h for protein extraction. Total proteins were extracted from inoculated leaves of Ov-NahG plants with W + OS treatments as described (Jongsma 1994). Bradford method (Bradford 1976) was used to determine the protein content of the samples. TPI activities were analyzed with the radial diffusion assay described (Jongsma 1993).

2.5 Immunoblot analysis

Total proteins from leaves of transiently over-expressed NaMLP or EV were extracted as described previously (Jongsma 1994). Three protein extract replicates were separated in 10% SDS-PAGE and transferred to immobilon PVDF membrane (Millipore Corporation) by using the Semi-Dry Transfer Cell system (Bio-Rad). Immuno-reacting proteins were detected by anti-GFP/Actin (1:5000, Abmart Corporation) as the primary antibody and peroxidase-conjugated goat anti-rabbit/goat anti-mouse IgG (1:20,000; Proteintech Corporation) as the secondary antibody. The results were imaged by MicroChemi 4.2 Bio Imaging Systems.

2.6 Real‑time PCR assay

Total RNA and cDNA and was extracted and synthesized as described (Wu, et al. 2013) Real-time PCR was performed as described by (Sun, et al. 2014) on a CFX Connect qPCR System (Bio-Rad) with iTaq Universal SYBR Green Supermix (Bio-Rad) and gene-specific primers (Supplementary Table). The unknown samples transcripts were quantified by the method as described (Song, et al. 2019) and the NaActin Ⅱ gene, which were unaltered and used as an internal standard (Xu, et al. 2018).

2.7 Generation of NaMLP‑silenced plants via virus induced gene silencing

Specific 366 bp of the NaMLP cDNA fragments were amplified by primers (CJ03 and CJ04) and cloned into pTV00, Agrobacterium tumefaciens (strain GV3101) cells carrying above constructs were mixed with those having pBINTRA, and inoculated into three leaves of 4-week-old N. attenuata plant, thus generating NaMLP-silenced (VIGS NaMLP) plants. Phytoene desaturase (PDS) was silenced to monitoring the progress of VIGS (Saedler and Baldwin 2004). When the leaves of NaPDS-silenced plants began to bleach, the 0 leaves of VIGS NaMLP plants and empty vector plants (EV plants) were selected and inoculated with A. alternata for further experiments. Around 30 plants were inoculated with each construct, and typically 24 biological replicates per construct exhibiting efficient silencing were used for each experiment. All the VIGS experiments were performed three times.

2.8 Generation of stable NaMLP‑silenced and over-expressed plants

We silenced NaMLP in N. attenuata by Agrobacterium-mediated transformation (Krügel, et al. 2002; Zhao, et al. 2021) using a pRESC8 vector containing a specific 395bp NaMLP fragment (with primers CJ13_F and CJ14_R) in an inverted-repeat orientation. Two independently transformed F2 lines, each harboring a single insertion were selected for all further experiments (irNaMLP lines 11 and 12).

We also generated N. attenuata plants with NaMLP over-expressed stably via Agrobacterium-mediated transformation (OvNaMLP line 2 and 3). The full-length cDNA of NaMLP with two hemagglutinin (HA: YPYDVPDYA) epitopes at its C-terminus was cloned into pCAMBIA1301 vector after the 35S promoter with the in-fusion technique (Clontech). N. attenuata plants (WT) were transformed by Agrobacterium tumefaciens with this construct according to (Krügel, et al. 2002; Zhao, et al. 2021). T1 seeds were screened for single T-DNA inserts (1:3 segregation of hygromycin resistance). Two independently transformed F2 lines (OvMLP line 2 and 3), each harboring a single insertion, were selected and used in this study.

2.9 Methyl jasmonate (MeJA) and ethephon treatments

Methyl jasmonate (MeJA) treatments were conducted as described by (Xu, et al. 2018). When applied, the chemical is quickly de-methylated into jasmonic acid by plant MeJA-esterase (Wu, et al. 2008). A solution of 1 mM MeJA or 5 mM ethephon was prepared by distilled water, and sprayed with a fine mist on the 0 leaves of rosette-staged N. attenuata plants (32-day-old) and immediately covered with a plastic bag on the treated plant, sampled after the above process at 1 and 3 h, the plants sprayed with distilled water were used as control treatments.

3.1 NaMLP can be strongly induced by A. alternata while it is not essential for A. alternata resistance in N. attenuata

A miraculin-like protein gene (NaMLP, XM_019373761), which shared 34.18% amino acid identity to miraculin in Synsepalum dulcificum, was found highly up-regulated in N. attenuata after A. alternata inoculation. Transcriptome analysis (Song, et al. 2019) indicated that this gene was strongly up-regulated in N. attenuata leaves at 1 day post inoculation (dpi). To confirm this result, we also quantified NaMLP expression by real-time PCR. Compared with mock control, the expression of NaMLP was increased to 10.9-fold at 1 dpi, and nearly 388-fold at 3 dpi (Fig. 1a). Interestingly, A. alternata-elicited NaMLP was dramatically reduced in JA-deficient irAOC and ethylene-insensitive Ov-etr1 plants, suggesting that A. alternata-induced NaMLP is regulated by both JA and ethylene (Fig. 1b).

We studied the subcellular location of NaMLP by transient over-expression of NaMLP fused with GFP in N. benthamiana leaves. Our results showed that most of the signals were from cell membranes (Fig. 1c). To rule out the possibility of cell wall localization, we also transformed NaMLP::GFP to the protoplasts of N. attenuata. Indeed, strong signals were detected in cell membranes again (Fig. 1c), indicating NaMLP is a cell membrane protein.

To investigate the role of NaMLP during A. alternata infection, we generated plants stably silenced with NaMLP (irNaMLP line 11 and 12). Real-time PCR indicated that A. alternata-induced NaMLP expression was reduced by 94% in irNaMLP line 11, and 89% in irNaMLP line 12 at 1 dpi (Fig. 2a). When the source-sink transition leaves (0 leaves) of irNaMLP and wild-type (WT) were collected and inoculated with A. alternata for 7 d. No significant difference of lesion diameter were observed in two independent experiments (Fig. 2b), suggesting that NaMLP does not play an essential role in N. attenuata against A. alternata.

3.2 NaMLP can be specifically induced by oral secretion (OS) of S. litura and is required for the resistance of S. litura in N. attenuata

Previously, JA signaling has been demonstrated to be an essential signal for both A. alternata and herbivore resistance (Paschold, et al. 2008; Sun, et al. 2014). As A. alternata-induced NaMLP was dependent on JA signaling, but we found that it was not required for this pathogen resistance, we thus speculated that it might be involved in herbivore resistance.

To test whether the saliva of S. litura can specifically induce the expression of NaMLP, we detected its expression in the 0 leaves of rosette-staged plants, by wounding with a fabric pattern wheel and immediately applying with diluted OS of S. litura (Wounding + OS; W + OS) to simulate herbivory, or water (Wounding + Water; W + W) as a control. The expression of NaMLP was gradually but significantly increased after time with W + W treatments at all the time points from 0.5 h to 6 h (Fig. 3a). However, NaMLP transcripts were strongly amplified by W + OS treatments at all these time points (Fig. 3a). These results suggested that NaMLP could be specifically induced by the OS of S. litura.

Next, we tested whether NaMLP is required for herbivore resistance by growing S. litura larvae on WT and NaMLP-silenced plants generated via VIGS. Our results showed that larvae gained significantly more mass on VIGS NaMLP plants indicating that NaMLP is required for the resistant to S. litura (Fig. 3b).

We also tested caterpillar performance in plants stably silenced with NaMLP and plants over-expressed with NaMLP (OvNaMLP line 2 and 3). Compared with WT, Os-elicited NaMLP expression were significantly increased to 4.5-fold in Ov-NaMLP line 2, and 5-fold in OvNaMLP line 3 after W + OS 6 h treatments (Fig. 3c). Our results showed that NaMLP was required for N. attenuata resistance to S. litura, as the insect larvae performed better in NaMLP-silenced plants (irNaMLP line 11 and 12) while performed the worst in both lines over-expressed with NaMLP (Fig. 3d; Supplementary Figure). These results strongly suggest that NaMLP can be specifically induced by OS and confers N. attenuata resistance to S. litura larva.

3.3 NaMLP is a new KTI exhibiting TPI activity

To understand the reason why NaMLP conferred the resistance to S. litura, we performed protein sequence blast in Gene Bank. Homologs were found in Solanaceae plants, with the highest similarity proteins (with 93–100% identity) in Nicotiana species. Interestingly, several conserved reaction sites of KTI were identified in NaMLP. Thus, NaKTI2 and NtKTI, two KTIs identified previously, were selected for sequence alignment with NaMLP. The protein sequences of NaKTI2 exhibited 53.55% similarities to NtKTI, while NaMLP only exhibited 33.16% similarities to NtKTI (Fig. 4). Yet, NaMLP had typical structural features of KTIs, including trypsin inhibitor domain (Kunitz legume family PF00197 in Pfam database), two disulphide bonds, a single reactive site and a number of β-trefoil folds (Fig. 4).

Although NaMLP showed lower sequence similarity to NaKTI2, the fact that NaMLP had conserved reaction sites of KTIs lead us hypothesized that NaMLP is a KTI with TPI activity. We transiently over-expressed NaMLP-eGFP under the control of CaMV 35S promoter in Ov-NahG leaves. Western blot analysis by using GFP antibody revealed that the NaMLP-eGFP protein was highly expressed in three Ov-NahG leaves independently transformed with 35S::NaMLP-eGFP, but was not present in leaves transformed with EV (Fig. 5a). Our results showed that the TPI activities of crude protein increased nearly 6.8-fold in Ov-NahG leaves over-expressed with NaMLP-eGFP when compared with those leaves transformed EV (Fig. 5a).

We next investigated the TPI activities in leaves of WT and NaMLP VIGS plants at 3 dpi of A. alternata. TPI activity were significantly reduced in 0 leaves of VIGS plants when NaMLP was successfully silenced (Fig. 5b).

We also investigated the OS-elicited TPI activities in leaves of irNaMLP and OvNaMLP plants. Our results showed that after treated with W + OS 3 d, significantly lower levels of TPI activity were detected in 0 leaves of both irNaMLP lines of 11 and 12, but higher levels were in OvNaMLP line 2 and 3 (Fig. 5c). Thus, our data showed that NaMLP has not only typical KTI structural features, but also confers N. attenuata resistance to S. litura larva through TPI activities.

3.4 Synergetic induction of NaMLP by JA and ethylene.

As A. alternate-induced NaMLP expression was significantly reduced in irAOC and Ov-etr1 plants (Fig. 1b), we speculate that Os-elicited NaMLP may also be regulated by JA and ethylene. Indeed, the induction of NaMLP expression by OS of S. litura was dramatically reduced in ethylene-reduced plants (irACO) and JA-deficient irAOC plants (Fig. 6a).

Since the induction of the NaMLP is dependent on the JA and ET signaling, we next investigated whether exogenously applied MeJA and ethephon could also induce the expression of the NaMLP. NaMLP transcripts were highly induced by MeJA or ethephon. The expression levels of NaMLP could reach to 886-fold that of the control after 3 h of MeJA treatment, and could reach nearly 26-fold after ethephon treatments (Fig. 7a). Interestingly, co-treatment with MeJA and ethephon for 3 h led to a much higher induction of NaMLP to 11235-fold (Fig. 7a).

Unlike NaMLP, Os-elicited NaPI was solely dependent on JA as it was only dramatically reduced in JA-deficient irAOC plants but not in ethylene-reduced irACO or WRKY3-silenced plants (Fig. 6b). When treated with MeJA or ethephon, NaPI could only respond to MeJA at both 1 and 3 h (Fig. 7b). In terms of NaKTI2, OS-elicited NaKTI2 was largely reduced in WRKY3-silenced plants, but only slightly reduced JA-deficient irAOC plants (Fig. 6c). When treated with MeJA or ethephon, neither treatment could induce NaKTI2 expression (Fig. 7c).

To further confirm the synergistic induction of the NaMLP by MeJA and ethephon is dependent on endogenous JA and ET signaling, 0 leaves of WT, irAOC, and irCOI1 plants were co-sprayed with MeJA and ethephon. NaMLP was significantly induced in WT and irAOC, but not in irCOI1 plants under the co-application of MeJA and ethephon (Fig. 8a), suggesting that endogenous JA perception is required for the synergistic induction of NaMLP. Similarly, in the presence of MeJA and ethephon, the NaMLP was significantly induced in WT and irACO, but not in Ov-etr1 plants (Fig. 8b), suggesting that endogenous ethylene signaling transduction is also required for the synergistic induction of NaMLP.

Thus, our data strongly indicates that N. attenuata plants accumulated different PIs through different signaling after herbivory, and NaMLP is synergistically induced by JA and ethylene signaling in N. attenuata plants when attacked by insect herbivores.

NaMLP was not required for A. alternata resistance but required for herbivore resistance in N. attenuata

Miraculin is known for its property of transforming sourness into sweetness (Mohammad, et al. 2011). Several miraculin-like proteins (MLPs) are reported to be involved in pathogen resistance. RlemMLP2 could inhibit the growth of Alternaria spores in rough lemon (Tsukuda, et al. 2006). In N. attenuata, NaMLP was highly elicited after A. alternata infection (Fig. 1). However, our data did not support that NaMLP played an essential role in A. alternata resistance, as no significant difference of lesion diameter were observed in WT and both NaMLP-silenced lines (Fig. 2).

Instead, our results strongly indicated that NaMLP conferred herbivore resistance. NaMLP transcripts responded strongly and specifically to the OS of S. litura (Fig. 3). Importantly, larvae grown better on plants silenced with NaMLP with both virus induced-gene silencing and stable transformation with RNAi construct (Fig. 3). In addition, we also generated stable transformation lines with NaMLP over-expressed, and S. litura larvae performed the worst in these plants comparing to WT and NaMLP-silenced plants (Fig. 3). Similar cases that MLP was involved in herbivore resistance were also found in curry tree (Gahloth, et al. 2011) and eggplant (Lopez-Galiano, et al. 2017).

NaMLP is a new KTI, exhibiting TPI activities

MKMLP in curry tree has the TPI activities to inhibit the activity of intestinal protease of Helicoverpa armigera and S. litura (Gahloth, et al. 2011). In eggplant, MLP can inhibit the activity of protease in the intestinal juice of potato beetle larvae (Lopez-Galiano, et al. 2017). However, it is not clear whether NaMLP has TPI activities.

Sequence blast and alignment analysis showed that although NaMLP has low sequence similarity to NtKTI, it has typical KTI domains, including two disulphide bonds, a single reactive site, a number of β-trefoil folds, and the trypsin inhibitor domain (Oliva et al. 2010; Bendre et al. 2018). Further analysis did show that NaMLP has TPI activities. When NaMLP was over-expressed, no matter transiently in Ov-NahG or stably in N. attenuata, plant leaves had higher TPI activities (Fig. 5). However, when NaMLP was silenced via VIGS or stable transformation lines with RNAi constructs, plant leaves had lower TPI activities (Fig. 5).

Thus, our study revealed that NaMLP is a new KTI with typical KTI structural features, and required for N. attenuata resistance to S. litura larva through its TPI activity.

NaMLP, a new TPI regulated synergistically by JA and ethylene in Nicotiana attenuata

Upon insect herbivore attack, host plants will usually accumulate large amount of TPIs to suppress the digestive machinery of insects. Thus such inhibitors were considered as an important part of plant defense arsenal against herbivores.

Currently, two different inhibitors, including NaPI and NaKTI2 were found in wild tobacco N. attenuata after herbivory. NaPI, which belongs to potato PI-II family with 77 known PI-II repeat domains, was demonstrated to be an effective defense against hornworm Manduca sexta (Zavala, et al., 2004). OS-elicited NaPI was strongly reduced in JA-deficient irAOC plants but not in WRKY3-silenced plants or ethylene-reduced irACO plants (Fig. 6), indicating that JA signaling played a key role in OS-elicited NaPI. This result was further supported by the strong elicitation of NaPI by exogenous MeJA treatments (Fig. 7). However, supplied ethephon had not effect on NaPI expression (Fig. 7). NaKTI2 was a Kunitz trypsin inhibitor gene recently identified as a defense gene against S. litura (Yin, et al. 2021). Unlike NaPI, OS-elicited NaKTI2 was mainly depended on transcription factors NaWRKY3 and NaWRKY6 (Yin, et al. 2021). NaKTI2 transcripts were not altered after MeJA or ethephon treatments (Fig. 7).

In this study, we revealed a new TPI after herbivory, NaMLP. The expression of this gene is controlled by both JA and ethylene pathways in a synergistic way. OS-elicited NaMLP was strongly reduced in both JA-deficient irAOC plants and ethylene-reduced irACO plants but not in WRKY3-silenced plants (Fig. 6). Furthermore, NaMLP transcripts could be significantly elicited by exogenous MeJA or ethephon treatments alone, but it levels were dramatically amplified when treated with MeJA and ethephon simultaneously, suggesting a synergistic induction of NaMLP by JA and ethylene pathways.

Thus, we concluded that once the leaves of N. attenuata were treated with OS, three signaling were activated, including JA, ethylene and WRKY3, which in turn regulated NaPI expression through JA signaling, NaKTI2 via WRKY3, and NaMLP by JA and ethylene in a synergistic way; finally all three TPIs, including NaPI, NaKTI2 and NaMLP, contributed to the OS-elicited TPI activities and to herbivore resistance (Fig. 9). The findings in this study elucidate a new source of TPI activities, and provide new insights to the complexity of the regulation of TPI-based defense.

Collectively, our results demonstrated that NaMLP is a new KTI coding gene regulated by JA and ethylene in a synergistic way, and confers S. litura resistance in N. attenuata.

ACKNOWLEDGMENTS

We thank Prof. Ian T. Baldwin (Max-Planck Institute for Chemical Ecology, Jena, Germany) for providing the transgenic N. attenuata seeds irAOC, irACO, irCOI1, and Ov-etr1, and the Service Center for Experimental Biotechnology of Kunming Institute of Botany, the Chinese Academy of Sciences (CAS), for plant growth support. This project was supported by the National Science Foundation of China (NSFC Grant No. 31670262) to Prof. Jinsong Wu.

AUTHOR CONTRIBUTIONS

Jinsong Wu conceived and designed the project; Jinsong Wu and Mao Yang prepared the manuscript; Junbin Cheng cloned NaMLP and performed VIGS experiments, tested TPI activities of NaMLP in VIGS plants and Ov-NahG plants, and found the synergistic induction of NaMLP by JA and ethylene; Min Yin generated RNAi plants and found the different regulation of TPIs; Mao Yang screened stable over-expression plants and performed caterpillar performance. All authors contributed to manuscript revision, read and approved the submitted version.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

Bhattacharyya A, Leighton SM, Babu CR (2007) Bioinsecticidal activity of Archidendron ellipticum trypsin inhibitor on growth and serine digestive enzymes during larval development of Spodoptera litura. Comp Biochem Phys C 145:669-677
Bradford (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 (1):248-254. .
Chen C-l, Yuan F, Li X-y, Ma R-c, Xie H (2021) Jasmonic acid and ethylene signaling pathways participate in the defense response of Chinese cabbage to Pectobacterium carotovorum infection. Journal of Integrative Agriculture 20:1314-1326
Eberl F, Fabisch T, Luck K, Kollner TG, Vogel H, Gershenzon J, Unsicker SB (2021) Poplar protease inhibitor expression differs in an herbivore specific manner. BMC Plant Biol 21:170
Gahloth D, Shukla U, Birah A, Gupta GP, Kumar PA, Dhaliwal HS, Sharma AK (2011) Bioinsecticidal activity of Murraya koenigii miraculin-like protein against Helicoverpa armigera and Spodoptera litura. Arch Insect Biochem Physiol 78:132-144
Halitschke R, Baldwin IT (2003) Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J 36:794-807
Jimenez T, Martin I, Labrador E, Dopico B (2007) A chickpea Kunitz trypsin inhibitor is located in cell wall of elongating seedling organs and vascular tissue. Planta 226:45-55
Jongsma BP, Stiekema WJ (1993) Quantitative determination of serine proteinase inhibitor activity using a radial diffusion assay. Analytical Biochemistry 212 (1):79-84. .
Jongsma BP, Visser B, Stiekema WJ ( 1994) Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack, wounding and virus infection. Planta 195 (1):29-35.
Krügel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12:177-183
Li J, Wu J (2016) Scopolin, a glycoside form of the phytoalexin scopoletin, is likely involved in the resistance of Nicotiana attenuata against Alternaria alternata. J Plant Pathol 98:641-644
Lopez-Galiano MJ, Ruiz-Arroyo VM, Fernandez-Crespo E, Rausell C, Real MD, Garcia-Agustin P, Gonzalez-Bosch C, Garcia-Robles I (2017) Oxylipin mediated stress response of a miraculin-like protease inhibitor in Hexanoic acid primed eggplant plants infested by Colorado potato beetle. J Plant Physiol 215:59-64
Mohammad, Jin SB, Park JW, Boo KH, Sun HJ, Kim YW, Lee HY, Riu KZ, Kim JH (2011) Functional Expression of Miraculin, a Taste-modifying Protein, in Transgenic Miyagawa Wase Satsuma Mandarin (Citrus unshiu Marc.). J Korean Soc Appl Bi 54:24-29
Paschold A, Bonaventure G, Kant MR, Baldwin IT (2008) Jasmonate perception regulates jasmonate biosynthesis and JA-Ile metabolism: the case of COI1 in Nicotiana attenuata. Plant & cell physiology 49:1165-1175
Paschold A, Halitschke R, Baldwin IT (2007) Co(i)-ordinating defenses: NaCOI1 mediates herbivore- induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses. Plant J 51:79-91
Saedler R, Baldwin IT (2004) Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata. J Exp Bot 55:151-157
Song N, Ma L, Wang W, Sun H, Wang L, Baldwin IT, Wu J (2019) An ERF2-like transcription factor regulates production of the defense sesquiterpene capsidiol upon Alternaria alternata infection. J Exp Bot 70:5895-5908
Sun H, Hu X, Ma J, Hettenhausen C, Wang L, Sun G, Wu J, Wu J (2014) Requirement of ABA signalling-mediated stomatal closure for resistance of wild tobacco to Alternaria alternata. Plant Pathology 63: 1070-1077
Sun H, Song N, Ma L, Li J, Ma L, Wu J, Wu J (2017) Ethylene signalling is essential for the resistance of Nicotiana attenuata against Alternaria alternata and phytoalexin scopoletin biosynthesis. Plant Pathology 66:277-284
Sun H, Wang L, Zhang B, Ma J, Hettenhausen C, Cao G, Sun G, Wu J, Wu J (2014) Scopoletin is a phytoalexin against Alternaria alternata in wild tobacco dependent on jasmonate signalling. Journal of experimental botany 65:4305-4315
Tsukuda S, Gomi K, Yamamoto H, Akimitsu K (2006) Characterization of cDNAs encoding two distinct miraculin-like proteins and stress-related modulation of the corresponding mRNAs in Citrus jambhiri lush. Plant Mol Biol 60:125-136
Wu J, Wang L, Baldwin IT (2008) Methyl jasmonate-elicited herbivore resistance: does MeJA function as a signal without being hydrolyzed to JA? Planta 227:1161-1168
Wu J, Wang L, Wunsche H, Baldwin IT (2013) Narboh D, a respiratory burst oxidase homolog in Nicotiana attenuata, is required for late defense responses after herbivore attack. J Integr Plant Biol 55:187-198
Xu Z, Song N, Ma L, Fang D, Wu J (2018) NaPDR1 and NaPDR1-like are essential for the resistance of Nicotiana attenuata against fungal pathogen Alternaria alternata. Plant Divers 40:68-73
Yin M, Song N, Chen S, Wu J (2021) NaKTI2, a Kunitz trypsin inhibitor transcriptionally regulated by NaWRKY3 and NaWRKY6, is required for herbivore resistance in Nicotiana attenuata. Plant Cell Rep 40:97-109
Zavala JA, Patankar AG, Gase K, Hui D, Baldwin IT (2004) Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiol 134:1181-1190
Zhao L, Li R, Wu J (2021) Optimization of rooting conditions in tissue-cultured seedlings of Nicotiana attenuata and its applications in gene transformation. Subtropical Plant Science 50:175-181

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published 06 Feb, 2023

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NaMLP, a new identified Kunitz trypsin inhibitor regulated synergistically by JA and ethylene, confers Spodoptera litura resistance in Nicotiana attenuata

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Abstract

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1. Introduction

2. Materials And Methods

2.1 Plant and fungal material

2.2 S. litura performance and leaf treatments

2.3 Sequence analysis

2.4 Transient over-expression and trypsin protease inhibitor (TPI) activity assay

2.5 Immunoblot analysis

2.6 Real‑time PCR assay

2.7 Generation of NaMLP‑silenced plants via virus induced gene silencing

2.8 Generation of stable NaMLP‑silenced and over-expressed plants

2.9 Methyl jasmonate (MeJA) and ethephon treatments

3. RESULTS

3.1 NaMLP can be strongly induced by A. alternata while it is not essential for A. alternata resistance in N. attenuata

3.2 NaMLP can be specifically induced by oral secretion (OS) of S. litura and is required for the resistance of S. litura in N. attenuata

3.3 NaMLP is a new KTI exhibiting TPI activity

3.4 Synergetic induction of NaMLP by JA and ethylene.

4. Discussion

NaMLP was not required for A. alternata resistance but required for herbivore resistance in N. attenuata

NaMLP is a new KTI, exhibiting TPI activities

NaMLP, a new TPI regulated synergistically by JA and ethylene in Nicotiana attenuata

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