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

We identified a miraculin-like protein (NaMLP) who is a new Kunitz trypsin inhibitor regulated synergistically by JA and ethylene signals and confers Spodoptera litura resistance in wild tobacco Nicotiana attenuata. The findings revealed a new source of trypsin inhibitor activities after herbivory, and provide new insights into the complexity of the regulation of trypsin inhibitor-based defense after insect herbivore attack. Upon insect herbivore attack, wild tobacco 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 or overexpression of NaMLP had no effect on the 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 overexpressed 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 overexpressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP overexpressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficient AOC silencing and ethylene-reduced ACO-silencing 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 pathways in a synergistic way.


Introduction
Plant protease inhibitors (PIs) have been known 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 arsenal 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 1 3 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 defend against insect herbivores.
Plant hormones play key regulatory roles in plant defense responses, especially JA and ethylene signaling . 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 expression was completely dependent on JA signaling, since it was abolished in JA-deficient irAOC plants (Yin et al. 2021). However, transcripts of NaKTI2 were only slightly regulated by JA signaling, but mainly dependent on transcription factor NaW-RKY3 and NaWRKY6 (Yin et al. 2021).
Alternaria alternata is a notorious necrotrophic fungus causing brown spot disease in Nicotiana species. When infected by A. alternata, N. attenuata plants will accumulate certain amounts of phytoalexins, capsidiol, scopoletin, and scopolin to defend against this pathogen (Li and Wu 2016;Sun et al. 2014a, b;Song et al. 2019). 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. 2014a(Sun et al. , b, 2017. 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 was strongly induced in N. attenuata after A. alternata infection drew our attention. By generating NaMLP-silenced and overexpressed plants, we found that NaMLP was required for resistance against S. litura instead of A. alternata. Further sequence alignment and functional tests uncovered that NaMLP was a new KTI. Finally we demonstrated that NaMLP was a KTI regulated synergistically by JA and ethylene in N. attenuata.

Plant and fungal material
A 31st generation of inbred line of N. attenuata was used as the wild type (WT). Stably transformed lines of JAdeficient 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), overexpressed 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. 2014a, b).

S. litura performance and leaf treatments
S. litura eggs were purchased from Keyun biological company and hatched in a growth chamber at 26 °C for 16-h light and 24 °C 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 at indicated time points. 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 sourcesink 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).

Transient overexpression and trypsin protease inhibitor (TPI) activity assay
The open reading frame of NaMLP was amplified by primers (CJ09 and CJ10) and insert directly at N-terminal of eGFP under the control of CaMV 35S promoter in pCAM-BIA1301. 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 et al. 1994). Bradford method (Bradford 1976) was used to determine the protein content of the samples.
TPI activities were analyzed with the radial diffusion assay (Jongsma and Stiekema 1993). In brief, 1 µg/mL trypsin (42 nM) was prepared in agar plates. The trypsin will digest N-acetyl-DL-phenylalanine-β naphthylester (APNE, Sigma) to produce dark red color. Dilution series of soybean TPI (Sigma) were used as standards to plot a reference curve according to the concentrations of soybean TPI and diameters of inhibition zones where the digestion of APNE by trypsin is inhibited by TPI. The TPI activities of unknown samples from N. attenuata leaves after OS treatments were calculated from the standard curve by using the diameters of the inhibition zone, and expressed as nanomole per milligram of total protein.

Immunoblot analysis
The total proteins from leaves of transiently overexpressed NaMLP or EV were extracted as described previously (Jongsma et al. 1994). Three protein extract replicates were separated in 10% SDS-PAGE and transferred to immobilon PVDF membrane (Millipore Corporation) 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 antirabbit/goat antimouse IgG (1:20,000; Proteintech Corporation) as the secondary antibody. The results were imaged by MicroChemi 4.2 Bio Imaging Systems.

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. 2014a, b) on a CFX Connect qPCR System (Bio-Rad) with iTaq Universal SYBR Green Supermix (Bio-Rad) and gene-specific primers (Supplementary Table). The transcripts of unknown samples were quantified by the method as described (Song et al. 2019) and the NaActin II gene, whose expression was unaltered, was used as an internal standard (Xu et al. 2018).

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.

Generation of stable NaMLP-silenced and overexpressed plants
We silenced NaMLP in N. attenuata by Agrobacteriummediated transformation (Krügel et al. 2002;Zhao et al. 2021) using a pRESC8 vector containing a specific 395 bp 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 overexpressed 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.

Methyl jasmonate (MeJA) and ethephon treatments
Methyl jasmonate (MeJA) treatments were conducted as described by (Xu et al. 2018). When applied, the chemical 1 3 is quickly de-methylated into jasmonic acid by plant MeJAesterase (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.

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. When 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 signaling pathways (Fig. 1b).
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 differences of lesion diameter were observed in two independent experiments (Fig. 2b). We also did not observed significant differences of lesion diameter developed in NaMLP-overexpression line 2 when compared to those in WT (Supplementary Fig. 1). All these results suggested that NaMLP does not play an essential role in N. attenuata resistance against A. alternata.

NaMLP can be specifically induced by oral secretion (OS) of S. litura and is required for resistance to S. litura in N. attenuata
Previously, JA signaling was demonstrated to be an essential signal for both A. alternata and herbivore resistance (Paschold et al. 2008;Sun et al. 2014a, b). Although A. alternata-induced NaMLP was dependent on JA signaling, we found that it was not required for this pathogen resistance, thus we 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 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. Fig. 1 Elicitation of NaMLP transcripts by A. alternate. a NaMLP transcripts were measured by real-time PCR in the source-sink transition leaves (0 leaves) of WT plants treated with mock or with A. alternata at 1, and 3 days post inoculation (dpi). All transcriptional levels were normalized with a housekeeping gene NaActin II. Values are means ± SE for five biological replicates. Asterisks indicate the level of significant difference between WT plants with the different treatments (Student's t test: ***p < 0.005). b NaMLP transcripts were measured by real-time PCR in 0 leaves of WT, irAOC (JA deficient) and Ov-etr1 (ethylene insensitive) plants treated with mock or with A. alternate at 3 days post inoculation (dpi). Asterisks indicate the level of significant difference between WT plants with the different treatments (Student's t test: ***p < 0.005) Next, we tested whether NaMLP is required for herbivore resistance by growing S. litura larvae on WT and NaMLPsilenced plants generated via VIGS. Our results showed that larvae gained significantly more mass on VIGS NaMLP plants after 10 d, 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 overexpressed 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 fivefold 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 overexpressed with NaMLP ( Fig. 3d; Supplementary Fig. 2). These results strongly suggest that NaMLP can be specifically induced by OS and confers N. attenuata resistance to S. litura larva.

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 disulfide bonds, a single reactive site and a number of β-trefoil folds (Fig. 4).
Although NaMLP shows lower sequence similarity to NaKTI2, the fact that NaMLP has conserved reaction sites of KTIs lead us hypothesized that NaMLP is a KTI with TPI activity. We transiently overexpressed 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 overexpressed with NaMLP-eGFP when compared with those leaves transformed empty vector (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 3 d after W + OS treatments, 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.

Fig. 2
NaMLP is not essential for A. alternata resistance in N. attenuate. a Mean (± SE) NaMLP transcripts were measured by real-time PCR in 0 leaves of four biological replicates in WT, irNaMLP line 11 and 12 after A. alternata inoculation at 1 dpi. Asterisks indicate the level of significant difference between WT and irNaMLP plants with the same treatments (Student's t test: *p < 0.05; ***p < 0.005). b Silencing NaMLP had no effect on lesion diameter developed on N. attenuata leaves after A. alternata inoculation at 7 dpi

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 speculated 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, and slightly by 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 11,235-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 confirm whether 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  (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 signals 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
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 NaMLP-silenced or overexpressed lines ( Fig. 2; Supplementary Fig. 1).
Some cases reported that MLPs were involved in herbivore resistance in curry tree (Gahloth et al. 2011) andeggplant (Lopez-Galiano et al. 2017). Our results also 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 either via VIGS or stable transformation with RNAi construct (Fig. 3). In addition, we also generated stable transformation lines with NaMLP overexpressed, and S. litura larvae performed the worst in these plants comparing to WT and NaMLP-silenced plants (Fig. 3).
What is the reason why NaMLP is highly induced by A. alternata but does not play a role in resistance against this fungus? Previously, we have shown that N. attenuata plants activate both JA and ethylene signaling pathways after A. alternata challenge (Sun et al. 2014a(Sun et al. , b, 2017. Similarly, insect herbivores also elicited both JA and ethylene signaling pathways in N. attenuata plants (Paschold et al. 2007;von Dahl et al. 2007). Thus, both insect herbivores and A. alternata will elicit similar defense responses, especially those shared in their regulation by both JA and ethylene signaling pathways. The elicitation of NaMLP serves a good example Fig. 4 Sequence alignment of NaMLP with known KTIs. Sequences were retrieved from the National Center for Biotechnology Information. NaMLP (N. attenuata, XP_019229306) were used for sequence alignment with Miraculin (Synsepalum dulcificum, BAH84844), NaKTI2 (N. attenuata, XP_019230715.1), and NtKTI (N. tabacum, ACL12055). Conserved and similar amino acid residues were shown with black and gray shading. The brackets indicated the disulfide bonds, triangles denote the Kunitz motif, a boxed region (full line) indicates the reactive site, and the arrows represented β-sheets of defense responses induced by both insect and pathogenic fungus through JA and ethylene signaling pathways. In this context, it will be very interesting to investigate whether plants will have higher defense responses to insect herbivore after A. alternata inoculation.

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 Fig. 5 NaMLP exhibits Trypsin protein inhibitor activity. a Western blot analysis (left) of protein extracts in leaves of Ov-NahG plants transiently overexpressed with NaMLP-eGFP or empty vector (EV). Three NaMLP-eGFP overexpressed samples were separated in lanes 1, 2 and 3, and EV samples in lanes 4. GFP antibody was used to detect NaMLP-eGFP (expected around 59 kDa, in the left panel). TPI activities (right) as detected by radial diffusion experiment of total proteins (three biological replicates) in overexpression of NaMLP-eGFP leaves. Asterisks indicate level of significant difference between EV and 35S::NaMLP-eGFP protein samples (Student's t test: ***p < 0.001). b A. alternate-induced NaMLP expression (lefte) and TPI activities (right) were detected in five biological replicates of 0 leaves of EV and VIGS NaMLP plants at 3 dpi. Asterisks indicate level of significant difference between EV and VIGS NaMLP (Student's t test: *p < 0.05; ***p < 0.001). c Mean (± SE) trypsin protein inhibitor activities were detected in 8 biological replicates of + 1 leaves of WT, stable NaMLP-silenced plants and stable NaMLP overexpressed plants. Asterisks indicate level of significant difference between EV/WT and transgenic samples with the same treatments (Student's t test: **p < 0.01; ***p < 0.001) 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 disulfide 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 overexpressed, no matter transiently in Ov-NahG or stably in N. attenuata, plant leaves had higher TPI activities (Fig. 5). On the other hand, when NaMLP was silenced via VIGS or stable transformation with RNAi constructs, plant leaves had lower TPI activities (Fig. 5), which is consistent with better performance of S. litura ( Fig. 3 and supplementary Fig. 2).
Thus, our study revealed that NaMLP is a new KTI with typical KTI structural features, and is required for N. attenuata resistance to S. litura larva through its TPI activity. Fig. 6 NaMLP, NaPI and NaKTI2 transcripts were measured by realtime PCR in the 0 leaves of WT, irACO, irAOC and irWRKY3 plants treated with mock or OS 3 h. Asterisks indicate level of significant difference between WT and transgenic plants with the same treatments (Student's t test: *p < 0.05; **p < 0.01; ***p < 0.001) Fig. 7 NaMLP, NaPI and NaKTI2 transcripts were measured by realtime PCR in five replicates of 0 leaves in WT plants treated with water (as control), MeJA and Ethephon individually and simultaneously at 1, and 3 h. Asterisks indicate the level of significant difference between control and treated leaves (Student's t test: *p < 0.05; ***p < 0.005)

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 JAdeficient 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 identified a new TPI, NaMLP that could be specifically induced by OS of S. litura. 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 after herbivory.
In Arabidopsis, PDF1.2 is one of the best known defense gene synergistically regulated by JA and ethylene signaling pathways during pathogen infection (Penninckx et al. 1998). ERF1 is proved to be a key element in integration of both signals for the regulation of PDF1.2 (Lorenzo et al. 2003). Later, JAZ proteins were reported to interact with EIN3/EIL1 to repress their function in regulation of ERF1 expression (Zhu et al. 2011). It's currently unknown how NaMLP is regulated by JA and ethylene signals. Whether NaERF1 and NaEIN3 are involved needs more investigation.
Thus, we concluded that once the leaves of N. attenuata were treated with OS, three signaling pathways 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 these three TPIs contributed to the OS-elicited TPI activities and to herbivore resistance (Fig. 9). Of course, it would be very interesting to investigate which PI genes contributed most to insect herbivore resistance in the future. Anyway, the findings in this study revealed a new source of TPI activities after herbivory, and provide new insights into the complexity of the regulation of TPI-based defense after insect herbivore attack.
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. Fig. 8 Mean (± SE) NaMLP transcripts were measured by real-time PCR in five biological replicates of 0 leaves in WT, irAOC (JA deficient), irCOI1 (JA insensitive), irACO (ethylene reduced) and Ov-etr1 (ethylene insensitive) plants co-treated with MeJA and ethephon at 24 and 72 h. Asterisks indicate the level of significant difference between each genotypes co-treated with MeJA and ethephon (Student's t test: *p < 0.05; ***p < 0.005) Fig. 9 Working model of TPI activities elicited by S. litura oral secretions in N. attenuata. When the leaves of N. attenuata are attacked by insect herbivores, three independent signaling are activated, including JA, ethylene and transcription factor NaWRKY3. NaPI is regulated solely by JA signaling, while NaKTI2 is mainly by NaW-RKY3, and NaMLP is controlled by JA and ethylene in a synergistic way; finally all three TPIs, including NaPI, NaKTI2 and NaMLP, contributed to the insect herbivore-elicited TPI activities, and confer herbivore resistance