Polyamines Attenuate Ethylene-Mediated Defense Responses to Abrogate Resistance to Botrytis cinerea in Tomato

Transgenic tomato ( Solanum lycopersicum ) lines over-expressing yeast spermidine synthase ( ySpdSyn ), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani , bacterial pathogen Pseudomonas syringae pv. tomato DC3000, and larvae of the chewing insect tobacco hornworm Manduca sexta . The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild type plants; however, responses to A. solani , P. syringae , or M. sexta were similar to the wild type plants. Exogenous application of ethylene precursors, S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea . The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense. Our data indicate a crosstalk between PA and ET in modulating responses to B. cinerea . SlACS expression was induced in WT leaves upon B. cinerea infection but attenuated in transgenic plants. Consistent with this observation was the reversion of host susceptibility by ACC, ET biosynthesis Taken together,


INTRODUCTION
A complex set of host and pathogen genetic factors determine the outcome of plant responses to pathogens. Necrotrophic fungi deploy a variety of virulence factors that assist in colonization of a wide range of host species (Groll et al., 2008). Botrytis cinerea, a necrotrophic fungal pathogen that infects over 200 plant species, is the causal agent of the gray mold disease, resulting in significant economic losses. Information on how plants combat necrotrophic pathogens such as B. cinerea and what signaling molecules are involved in such interactions is beginning to emerge (AbuQamar et al., 2008;Laluk et al., 2011). Host immune response to infection is mediated by diverse regulatory processes, of which, plant hormone functions have been studied extensively in relation to disease (Spoel and Dong, 2008;Bari and Jones, 2009;Pieterse et al., 2009) Polyamines (PAs) are polycationic, ubiquitous compounds that have essential functions in all organisms studied thus far involving regulation at both transcriptional and translational levels (Veress et al., 2000;Kasukabe et al., 2004;Yoshida et al., 2004;Alcázar et al., 2005;Igarashi and Kashiwagi, 2006;Srivastava et al., 2007;Mattoo and Handa, 2008;Handa and Mattoo, 2010). Putrescine (Put), spermidine (Spd) and spermine (Spm) are the three most prominent PAs in plants. Decarboxylation of ornithine by ornithine decarboxylase (ODC) or arginine by arginine decarboxylase (ADC) leads to the synthesis of Put, which is converted to Spd by Spd synthase (SpdSyn), and Spd, in turn, then converted to Spm by Spm synthase (SpmSyn;Nambeesan et al., 2008). In these reactions, both SpdSyn and SpmSyn enzymes use aminopropyl residues derived from decarboxylated S-adenosylmethionine (dcSAM) which is synthesized from Sadenosylmethionine (SAM) by SAM decarboxylase (SAMdc; Martin-Tanguy, 1997;Bouchereau et al., 1999;Mehta et al., 2002). SAM is also a substrate for 1aminocyclopropane-1-carboxylic acid (ACC) synthase, a reaction that generates ACC, the immediate precursor of ET (Fluhr and Mattoo, 1996). PAs are present in various cellular compartments such as vacuole, mitochondria, chloroplast as well as in cell wall fractions (Kaur-Sawhney et al., 2003). While both nuclear and cytoplasmic localization of ODC have been reported, the ADC pathway is predominantly localized to the chloroplast. Spd synthase has been reported to be localized both in the chloroplast and cytoplasm (Nambeesan et al., 2008).  (Walter et al., 1985). Also, decreases in PAs have been 7 observed in plant-fungal and plant-viral infections (Bakanashvili et al., 1987;Edreva, 1997

8
Tomato cultivar Ohio 8245 was used to generate transgenic lines expressing the ySpdSyn gene under the control of the CaMV35S promoter using Agrobacteriummediated transformation (Fig. 1A). Several independent T 0 plants transformed with this chimeric construct were selected for kanamycin resistance and screened for the presence of the ySpdSyn gene using PCR. Kanamycin-resistant and PCR-positive plants were then screened for transgene and immunoreactive protein expression using RNA-blot and immunoblot analyses, respectively, as described in the methods section. Based on these analyses of the T 0 transgenic lines, two independent transgenic lines C4 and C15 were selected for studies described in this manuscript (Fig. 1B). T 1 seedlings were grown and homozygous C4 and C15 plants were generated. Spd levels in the leaf tissue were 2.1and 1.3-fold higher in C4 and C15, respectively, compared to the wild type (WT) cultivar ( Fig. 1C; 0 hours post infection). However, the pattern of Put levels was inconsistent, being 1.2-fold higher in C4 and 1.3-fold lower in C15 compared to WT plants (Fig. 1C).
Spm levels were lower in leaves of both the transgenic lines compared to WT, but C4 plants had greatly reduced Spm content than the C15 plants. These data suggest that ySpdSyn was functional in tomato and led to higher Spd levels.

Leaf Polyamine Levels Increase during Botrytis Infection
To determine the relationship between polyamine (PA) content and disease response, changes in PA levels were quantified after inoculation with B. cinerea (Fig. 1C hpi, the lesion sizes in WT, C4 and C15 leaves treated with DFMO or CHA were of the same extent ( Fig. 3E) suggesting that inhibitors of PA biosynthesis decrease susceptibility of transgenic leaves to B. cinerea. Treatment with Spd significantly increased lesion diameter in WT leaves at 72 hpi which was similar to that seen in C4 and C15 plants ( Fig. 3F-H). Thus, these data support above results that elevated levels of Spd increase susceptibility of tomato to B. cinerea. a transcription factor required for activation of defense response genes (Solano et al., 1998;Guo and Ecker, 2004). As shown in Fig. 4C, a 6-and 4-fold increase in the expression of SlEIL1 occurred in C4 and C15 transgenic plants, respectively, by 24 dpi, which was not observed in WT. The expression pattern of SlEIL2 was variable: in the WT and C4 these increased similarly, 2.5-fold, upon infection but in C15 leaves they were suppressed by 1.3-fold during infection (Fig. 4D). The expression of SlERF1B increased ~ 59-fold in WT compared to a relatively moderate, 14-and 4-fold increase in C4 and C15 leaves, respectively, at 24 hpi, which is consistent with the disease susceptibility phenotypes of the transgenic lines (Fig. 4E). These results support our contention that impaired or altered expression of components of ET biosynthesis and signaling pathways account for enhanced susceptibility of high-Spd tomato plants to B. cinerea. Interestingly, transcripts of β-1,3-glucanase, another PR gene, increased in both the C4 and C15 transgenic plants upon B. cinerea infection but this did not induce resistance (Fig. 4F). It is therefore likely that higher PA levels may contribute to general perturbations of cellular homeostasis that could subsequently cause a general upregulation of PR proteins and disease susceptibility.  Fig. 5A-C). These data confirm that ET is an important factor in this interaction. With respect to plant growth responses, ET is known to produce a characteristic seedling growth response called the triple response (Guzmán and Ecker, 1990). In the presence of exogenous ACC such a response was, however, not observed with the transgenic C4 and C15 seedlings compared to WT  (Kunkel and Brooks, 2002). We, therefore, tested WT and transgenic tomato seed germination for altered hormone sensitivity. WT, C4 and C15 seeds were plated on medium containing methyl-JA (MeJA) or ABA, and their germination and/or root elongation were analyzed (Supplemental Fig. S2). Neither C4 nor C15 transgenic plants showed germination defects or differences in root elongation as compared to WT in medium containing either hormone, suggesting that increased Spd does not interfere with responses to ABA or MeJA.

Spd-Accumulating Transgenic Lines are Similar to WT in Responses to Alternaria solani, Pseudomonas syringae and Tobacco Hornworm
The C4 and C15 transgenic plants were also tested for responses to other virulent pathogens such as A. solani (the causal agent of early blight), P. syringae (the bacterial speck disease), as well as tobacco hornworm M. sexta. Inoculation with A. solani did not result in enhanced lesion development in transgenic C4 and C15 leaves compared to WT (Fig. 6A). Infiltration of leaves with P. syringae and subsequent analysis of bacterial growth revealed no significant changes in bacterial titer 3 days after infiltration (Fig. 6B). In plants exposed to tobacco-hornworm larvae, increased defoliation was not observed (data not shown) but slight, though insignificant, differences in larval weight between the WT and the transgenic leaves were apparent. The leaves of C15 transgenic line were relatively more inhibitory to larval growth (Fig. 6C).

Increase in Spd Levels does not Alter Responses to Oxidative Stress
The response of WT, C4 and C15 seedlings to oxidative stress was measured by determining seedling growth in the presence and absence of hydrogen peroxide (H 2 O 2 ).
As shown in Supplemental Fig. S2, seedling growth of all the three genotypes was lower in the presence of H 2 O 2 . However, no differences were apparent in root or shoot growth between the WT, C4 and C15 seedlings (Supplemental Fig. S2). Additionally, treatment of leaves with methyl viologen did not exhibit any difference in response amongst the three genotypes (Supplemental Fig. S3). We interpret these data to suggest that increased Spd in transgenic leaves does not alleviate oxidative stress caused by H 2 O 2 or methyl viologen.

DISCUSSION
We present molecular and pharmacological evidence suggesting that Spd plays a significant and specific role in tomato response to the necrotrophic fungal pathogen B. physiological changes in transgenic Arabidopsis, rice, and pear plants over-expressing SpdSyn and SAMdc genes (Kasukabe et al., 2004;Wen et al., 2008;Peremarti et al., 2009). Unsuccessful attempts to obtain stable transgenic potato plants expressing a potato SAMdc under the CaMV 35S promoter while SAMdc antisense lines led to a range of stunted phenotypes also support this contention (Kumar et al., 1996). PAs play important roles during meiosis, sporulation and cell division, and thus have been suggested to modulate fungal development. Depletion of PAs is lethal for fungi (Tabor, 1981;Rajam and Galston, 1985;Walters, 1995). Use of DFMO and CHA, inhibitors of ODC and SpdSyn proteins, respectively, were effective in mitigating the growth of various fungal species such as B. cinerea (Rajam and Galston, 1985;Saftner et al., 1997), Tilletia spp However, the altered PA homeostasis in transgenic leaves treated with PA biosynthesis inhibitors was enough to influence host physiology and reduce lesion due to infection.
Spd-mediated enhanced susceptibility of transgenic tomato leaves suggests an impaired host-signaling network that leads to weakened immune responses against B. cinerea infection. Depending on the plant-pathogen interaction, treatment with ET may enhance resistance (Esquerré-Tugayé et al., 1979;El-Kazzaz et al., 1983b;Marte et al., 1993), induce susceptibility, or have no effect (El-Kazzaz et al., 1983a;Brown and Lee, 1993;Thomma et al., 1999) these results support the interpretation that Spd-induced susceptibility to B. cinerea is due to its interference with ET biosynthesis (SlACS) and response (SlERF1B) pathways.

The effect of Spd on downstream ET signaling was investigated to understand
Spd-mediated alteration of ET-dependent resistance to B. cinerea. ET is sensed and bound by a family of membrane receptor proteins that activate CTR1, a negative regulator of the ET-signaling cascade. Downstream, this signaling cascade is facilitated by the transcription factors EIN2, EIN3 and paralogs, the EIN3-like proteins (EILs; Solano et al., 1998;Stepanova and Ecker, 2000;Guo and Ecker, 2004). Three functionally redundant homologs of Arabidopsis EIN3, SlEIL1-3, have been identified in tomato (Tieman et al., 2001). ERF1 is an early response gene that has been implicated in several necrotrophic pathogen responses (Lorenzo et al., 2003). In line with Spd and ET interaction being competitive in B. cinerea-tomato interactions, we found that ETresponsive pathway genes such as SlERF1B, but not SlEIL1 and SlEIL2, displayed reduced expression in both the transgenic lines compared to the WT (Fig. 4). SA signaling has been implicated in resistance of hydroponically-grown tomatoes to another necrotroph, A. solani. Exogenously applied SA induced systemic acquired resistance (SAR) in these tomatoes, which led to effective resistance against A. solani (Spletzer and Enyedi, 1999). Likewise, application of a chemical inducer of SAR such as benzothiadiazole conferred resistance to potato against A. solani (Bokshi et al., 2003). In this context, it is interesting that the transgenic C4 and C15 tomato plants were similar to the WT in their response to inoculation with the hemibiotroph, P. syringae or with A.

Genetic data in
solani. Previously, studies with Arabidopsis have suggested that the ET-mediated disease resistance may be pathogen-specific based on the type of the necrotroph since ein2-1 mutants were more susceptible to B. cinerea but not A. brassicicola (Thomma et al., 1999). Our studies are consistent with these findings. Therefore, observed expression of PR1 in ySpdSyn transgenic plants might be a reflection of increased susceptibility to Botrytis rather than being a marker of enhanced SA response. It is noted here that SA or polyamines (Spd or Spm) when applied to tomato slices leads to suppression of SlACS transcripts and ET biosynthesis (Li et al. 1992), as depicted for SlACS here. Our work also differentiates Spd involvement from that of Spm in B. cinerea pathogenesis (Gonzales et al., 2011) since the ySpdSyn transgenic plants are relatively deficient in Spm compared to WT and, notably, during B. cinerea infection Spm levels actually decreased in the WT. Collectively, these data suggest a negative effect of increased Spd levels on ethylene synthesis and/or signaling, which leads to higher susceptibility of tomato leaves to B. cinerea (summarized in Fig.7).

Plant Growth
Tomato (Solanum lycopersicum cv. Ohio 8245) plants were grown in plastic pots containing compost soil mix in a greenhouse with a photoperiod extended to 15 h under fluorescent lights (160 W mol -1 m -2 s -1 ) at a temperature of 24 ± 4°C.

Generation of Transgenic Plants
Transgenic lines expressing ySpdSyn gene driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter were generated. The ySpdSyn was amplified from a yeast genomic library using the forward primer: ScSpe3XhoF (5'GCCGCTCGAGATGGCACAAGAAATCACTCACCCAA3') and reverse primer: ScSpe3XbaR (5'GCCGTCTAGACTAATTTAATTCCTTGGCTGCCCAG3'), and cloned into the pGEM-T Easy vector system (Promega). The insert was excised using restriction endonucleases, Xho1 and XbaI, and cloned in the sense orientation between a CaMV35S promoter and the 3' end of a pea rbcS-E9 gene in pKYLX71 (Schardl et al., 1987). This construct was introduced into the disarmed Agrobacterium tumefaciens LBA4404 by chemical transformation. Agrobacterium strains harboring the chimeric constructs were used to transform cotyledons of tomato cv. Ohio 8245 (Tieman et al., 1992). Fifteen independent transgenic plants expressing ySpdSyn under the CaMV35S promoter were generated (Nambeesan et al., 2010). Based on the transcript and protein expression analysis of T 0 transgenic plants, two independent transgenic plants, C4 and C15, were selected and used for further studies. Seeds from T 1 seedlings were analyzed for selecting homozygous plants using PCR. Lines homozygous for the transgenes were selected after PCR analyses of seedlings from T 2 seeds and used in studies presented here.

Fungal and Bacterial Disease Assay
The B. cinerea strain BO5-10 was used for disease assays. Fungal culture and preparation of conidial spore suspension were as described previously (AbuQamar et al., 2006). diameter was determined (n=60). WT and transgenic leaves were inoculated with 300 mg/ml A. solani cultures and lesion diameter was calculated 7 days after inoculation.
Bacterial disease assays were done essentially as described (Mengiste et al., 2003). Fully expanded leaves of 6-week-old tomato plants were infiltrated with suspensions of the bacterial strain P. syringae (OD 600 = 0.001, ~5x10 5 CFU/ml in 10 mM MgCl 2 ). Bacterial growth was determined using leaf discs from infected leaves at 0 and 3 days after infection (DAI) as described (AbuQamar et al., 2008). Bacterial titer per leaf area was determined in uniform leaf discs using a hole-punch. Each experiment was performed in triplicate and two leaf discs were collected from C4, C15 and WT plants for each replicate.

Tobacco Hornworm Feeding Trials
Tobacco hornworm M. sexta eggs and an artificial diet for the larvae were purchased from Carolina Biological Supply Company (Burlington, North Carolina). As recommended by the supplier, eggs were hatched by incubation at 26°C. The artificial diet for the hatched larvae was continued for 3 days before transfer to detached leaves or whole tomato plants. For whole plant assay, four larvae weighing 9-11 mg each were placed on each of six 8-week old WT, C4 and C15 plants grown in the green house.
The average larval weight at the beginning of the feeding trial was 7-9 mg for detached leaf assay and 9-11 mg for whole plant assay. The insects were left to feed for one and two weeks for the detached leaf and whole plant assays, respectively, after which the larval weight was determined (Abu-Qamar et al., 2008).

RNA-Blot, and Quantitative and Semi-quantitative RT-PCR Analyses
Total RNA was extracted from frozen (in liquid nitrogen) tomato leaf tissues as described (Lagrimini et al., 1987). For RNA-blot analyses, RNA was separated on 1.2% formaldehyde agarose gels and blotted onto Hybond N + nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The probes were labeled with 32 P by random priming using a commercial kit (Sigma). Probe hybridization was performed as described (Church and Gilbert, 1984

Inhibitor Treatments
WT and transgenic tomato plants were grown for 6 weeks in the greenhouse using compost soil mix with a photoperiod of 16h/8h (day/night) at a day/night temperature of 23/18°C. For growth regulator treatments, leaves were clipped and the petiole was Quantification of PA was performed in triplicate with each replicate consisting of at least 3 leaves.

Ethylene Response Assays
Seeds were surface sterilized with 70% ethanol for 2 min followed by a treatment with 35% commercial bleach (5.25% [w/v] sodium hypochlorite) plus 0.1% Tween 20 for 30 min in solution, and finally thoroughly rinsed with water to remove the bleach. For the triple response assay, tomato seeds were cultured on MS medium and 0.8% agar with or without ACC and incubated in the dark for 6 days (Abu-Qamar et al., 2008).

Hormone Treatments
Seeds prepared as above were plated in vitro on the medium containing MS with 0.8% agar with or without 10 µM methyl-jasmonate (MeJA), or 2 µM ABA. Seed germination was recorded after 7 days of growth. For ABA transfer assay, seeds were initially germinated on MS media for 3 days and then transferred to MS media supplemented with or without 10 µM ABA and seedling growth was studied. Solutions of MJ and ABA used in the experiments reported here were prepared and used as described previously         Table 1: Sequences of forward (F) and reverse (R) primers used for gene expression studies shown in Figure 2C and Figure 4 Supplemental Figure S1. Expression of ySpdSyn does not alter the triple response of WT and T 2 homozygous C4 and C15 transgenic seedlings in the presence of exogenous ACC.
Seeds were germinated on MS medium with or without 1 µM ACC and incubated in the dark for 6 days before responses of seedling growth were recorded.