Constitutive expression of a novel antimicrobial protein, Hcm1, confers resistance to both Verticillium and Fusarium wilts in cotton

Fusarium and Verticillium wilts, two of the most important diseases in cotton, pose serious threats to cotton production. Here we introduced a novel antimicrobial protein Hcm1, which comprised harpin protein from Xanthomonas oryzae pv. oryzicola (Xoc), and the chimeric protein, cecropin A-melittin, into cotton. The transgenic cotton lines with stable Hcm1 expression showed a higher resistance to Verticillium and Fusarium wilts both in greenhouse and field trials compared to controls. Hcm1 enabled the transgenic cotton to produced a microscopic hypersensitive response (micro-HR), reactive oxygen species (ROS) burst, and caused the activation of pathogenesis-related (PR) genes in response to biotic stress, indicating that the transgenic cotton was in a primed state and ready to protect the host from pathogenic infection. Simultaneously, Hcm1 protein inhibited the growth of Verticillium dahliae (V. dahliae) and Fusarium oxysporum (F. oxysporum) in vitro. The spread of fungal biomass was also inhibited in vivo since the V. dahliae biomass was decreased dramatically in transgenic cotton plants after inoculation with V. dahliae. Together, these results demonstrate that Hcm1 could activate innate immunity and inhibit the growth of V. dahliae and F. oxysporum to protect cotton against Verticillium and Fusarium wilts.

DI was investigated on June 24, 2014 in Henan according to historical peak incidences. These results showed that the DIs of the three transgenic lines were reduced to 66.77%, 49.83%, and 67.99% compared to the parent W0 (Fig. 2a,c), indicating that expressing Hcm1 in cotton conferred resistance to Fusarium wilt in a field condition.
Resistance of Hcm1-transformed cotton plants to Verticillium wilt. Isolates of V. dahliae can be characterized as defoliating or non-defoliating pathotypes based on symptoms expressed in cotton plants with the disease 9 . The defoliating V. dahliae isolate V991 and non-defoliating V. dahliae isolate BP2 were used to assess the resistance of Hcm1-transformed cotton in greenhouse conditions. Foliar damage and vascular discoloration was observed in parent W0 plants at 10 and 15 days after inoculation with V991 and BP2, respectively. With the outbreak of the disease, the Hcm1-transformed plants had only a small number of chlorotic and necrotic spots and there was almost no plant death, whereas the parent W0 and susceptible variety, Junmian 1, plants showed common large chlorotic and necrotic areas in their leaves, and some plants eventually died (Fig. S1). The results  showed that the DIs of the three transgenic lines were significantly lower than that of the parent W0 after inoculation with V991 and BP2, revealing that Hcm1 improved cotton tolerance to defoliating and non-defoliating V. dahliae in greenhouse conditions (Fig. 3a). Field trials to assess the performance of the transgenic lines against Verticillium wilt were conducted in Henan and Xinjiang provinces, in China during the 2014 cotton-growing season. Disease surveys were conducted on September 5, 2014 in Henan province and August 27, 2014 in Xinjiang province, since the peak incidence of Verticillium wilt in the field generally occurs in early August to mid-September in China. The DIs of the three transgenic lines decreased from 36.95% to 58.15% and from 25.33% to 34.03% compared to parent W0 (Fig. 3b,c) in Henan and Xinjiang provinces, respectively. The mortality rates of Hcm1-transformed lines decreased from 65.70% to 74.93% and 61.72% to 70.25% in Henan and Xinjiang, respectively (Fig. 3d). However, the resistance of transgenic lines to Verticillium wilt in Xinjiang was weaker than that in Henan. The reason may be the differences in climate, geographical conditions, or physiologic races in soils between the two provinces. In addition to the DI, the agronomic performance of the transgenic lines, including the height, lint percentage, number of fruit branches, single boll weight, and lint yield, was significantly higher than that of the parent W0 ( Table 2). The lower DIs and mortality rates, and the better agronomic performance of Hcm1-transformed lines demonstrated that Hcm1 conferred cotton resistance to Verticillium wilt, and improved its agronomic traits in the disease nurseries.
ROS burst occurred and the PR genes were activated in Hcm1-transformed plants after inoculation with V. dahliae. An ROS burst occurred when leaves of hpa1 Xoo -transformed plants were inoculated with V. dahliae 14 . In the leaves of Hcm1-transformed line H213 and the parent W0 plants, a reddish-brown precipitate was observed after inoculation with V. dahliae, as detected by 3, 3′ -diaminobenzidine tetrahydrochloride (DAB) 37,38 . However, the DAB staining in leaves of H213 plants was markedly different from that in parent W0 plants (Fig. 4a). In order to accurately observe this difference, H 2 O 2 content was measured. The basal H 2 O 2 content was higher in the leaves of transgenic H213 plants than in leaves of the parent W0 plants prior to inoculation. After inoculation with V. dahliae, H 2 O 2 content gradually increased in leaves of the parent W0 plants, and peaked   at 8 hours (hr), while in H213 leaves, H 2 O 2 content was increased dramatically and appeared in two peaks at 1 hr and 5 hr (Fig. 4b). These results showed that the Hcm1 transgenic lines displayed an ROS burst in response to biotic stress. Harpins can activate the PR genes in plants 14,22 . GhHSR203J and GhHIN1, which are considered as marker genes for HR 20,21 , were up-regulated in the Hcm1-transformed line H213 and the parent W0 (Fig. 4c). NPR1, which is a key transcriptional regulator in plant defense responses involving multiple signaling pathways 39 , was up-regulated in transgenic line H213 after inoculation with V. dahliae. The marker genes of salicylic acid (SA) and nitric oxide (NO) signaling pathways 22,40 , GhPR1 and GhNOA1, were also significantly up-regulated in the transgenic line H213 compared to the parent W0 (Fig. 4d), showing that the PR genes were activated in Hcm1-transformed plants in response to biotic stress.
A microscopic hypersensitive response (micro-HR) was observed in transgenic line H213 after leaf and root inoculation with V. dahlia. Harpins can induce an HR in tobacco following infiltration of leaf panels 2 . No visible HR was observed in cotton expressing Hpa1 Xoo , but a micro-HR was detected in plants after inoculation with V. dahliae 14 . Leaves from transgenic line H213 and parent W0 plants 0-12 hr after inoculation with V. dahliae conidia suspension were stained with trypan blue, which selectively stains dead or dying cells 41 . Leaves inoculated with sterile water were used as a control. No trypan blue-stained cells were observed in leaves from H213 or W0 plants inoculated with water, or in parent W0 plants inoculated with V. dahliae (Fig. 5a). However, in leaves from H213 plants, trypan blue-stained cells representing a micro-HR were observed by stereoscope at 8 hr and 12 hr after inoculation with V. dahliae (Fig. 5a). Leaves from H213 and W0 plants after root inoculation with V. dahliae conidia suspension in greenhouse conditions were also used for micro-HR detection. Leaves inoculated with sterile water were used as a control. Trypan blue-stained cells were observed in leaves from H213 plants inoculated with V. dahliae but not in leaves from parent W0 plants inoculated with sterile water or V. dahliae or in H213 plants inoculated with sterile water, revealing that micro-HR occurred in Hcm1-transformed plants in response to biotic stress (Fig. 5b). These data indicate that a micro-HR occurred when the Hcm1-transformed plants suffered biotic stress.

Hcm1 effectively inhibits the spread of V. dahliae in cotton. Cecropin A-melittin can normally
inhibit pathogens infection when the Hcm1 protein exists in the plasma membrane. Moreover, recent studies have shown that host targets of harpins may be present in the plasma membrane 20,42,43 . Our unpublished data suggest that Hpa1 Xoc is located in the plasma membrane and nuclear membrane of plant cells. Therefore, whether the Hcm1 protein exists in the plasma membrane is very important for the function of Hpa1 Xoc and cecropin A-melittin from Hcm1. We fused the Hcm1 coding region in frame with the N-terminus of GFP coding region under the control of the CaMV35S promoter to examine the subcellular localization of Hcm1. Onion epidermal cells were separately transformed with either the 35S::Hcm1::GFP fusion or the 35S::GFP plasmid control by particle bombardment. GFP-specific fluorescence was found in the cell membrane and other parts of cells transformed with the 35S::Hcm1::GFP fusion (Fig. 6a I-III). When the cell wall and cell membrane were separated by treatment with 20% sucrose for 15 min, GFP fluorescence was observed in the membrane but not in the cell wall ( Fig. 6a IV-VI). GFP fluorescence was detected throughout control cells transformed with the 35S::GFP plasmid (Fig. 6a VII-IX). These results indicate that Hcm1 is present in the cell membrane when Hcm1 is expressed in plant cells.
The Hcm1 protein shows broad antimicrobial activity in vitro 22 . The ability of crude cell-free elicitor preparations (CFEPs) of Hcm1 and the Hcm1-transformed proteins, which were extracted from prokaryotic expression and the leaves of transgenic line H213 plants, respectively, to inhibit the growth of V. dahliae and F. oxysporum on potato dextrose agar (PDA) and complete medium (CM) plates was tested. Carbendazim, CFEPs and Hcm1-transformed proteins caused an obvious inhibition halo on PDA and CM plates inoculated with F. oxysporum. A 5-fold dilution of Hcm1-transformed proteins also inhibited the growth of F. oxysporum compared to controls (Fig. 6b and Fig. S2). Carbendazim, CFEPs, and Hcm1-transformed proteins also inhibited the mycelial growth of V. dahliae on PDA and CM plates, while a 5-fold dilution of Hcm1-transformed protein had no effect on the growth of V. dahliae ( Fig. 6b and Fig. S2).
In order to verify the antimicrobial activity of Hcm1 against V. dahliae in vivo, the strength of green fluorescence was observed in cotton plants inoculated with a V. dahliae strain V991 harboring the GFP gene , since V. dahliae harboring the GFP gene emits fluorescence in cotton tissues 44 . 15 days after inoculation with V. dahliae, the green fluorescent signal was observed in the leaves of parent W0 plants but not Hcm1-transformed plants (Fig. S3). Although the green fluorescent signal was observed in the rhizome connections of Hcm1-transformed and parent W0 plants, the green fluorescent signal in Hcm1-transformed plants was significantly weaker than in parent W0 plants (Fig. 6c). In addition to observing the fluorescent signal, the biomass of V. dahliae strain V991 in cotton plants was measured with qRT-PCR. Determination of the V. dahliae strain biomass showed significant differences between V. dahliae-inoculated Hcm1-transformed and parent W0 plants. The biomass of V. dahliae strain V991 in the roots, stems, and leaves of Hcm1-transformed plants was significantly lower than in parent W0 plants (Fig. 6d). These results revealed that the expression of Hcm1 reduced the biomass of V. dahliae in cotton plants. Hcm1, therefore, effectively inhibited the spread of V. dahliae in cotton.

Discussion
Hcm1 was effective at controlling Fusarium wilt and Verticillium wilt. Previous studies have shown that harpin, applied as a foliar spray or expressed in plants, confers resistance to pathogens 14,24,45,46 . The antimicrobial proteins, cecropin A-melittin, also effectively confer plants with a resistance to a broad spectrum of pathogens 33,34,35 . Resistance to tobacco mosaic virus, bacterial Ralstonia solanacearum, and fungal Magnaporthe oryzae infections can be improved by spraying Hcm1 protein on plants prior to inoculation with plant pathogens 22 . In the present study, Hcm1 was transformed into a susceptible cotton variety, W0. In greenhouse conditions, the Hcm1-transformed cotton lines were resistant to disease caused not only by the defoliating and non-defoliating isolates of V. dahliae, but also F. oxysporum. In the V. dahliae and F. oxysporum-inoculated field, the Hcm1transformed plants showed lower DIs and mortality rates compared to parent W0 plants (Figs 2 and 3). The lint yields, which are regarded as the most important agronomic measurement of cultivar performance, of Hcm1transformed and parent W0 plants were not significantly different when the plants were grown in non-infected soil in field conditions (Table 1). When planted in V. dahliae-infected soil, the lint yields of Hcm1-transformed plants were 20-77% higher in Henan province and 27-73% in Xinjiang province than parent W0 plants ( Table 2). The results indicate that Hcm1 is effective at controlling Fusarium and Verticillium wilts.
Hcm1 led to an ROS primed state for plant defense activation in cotton. Higher plants are capable of inducing some stress "memory" or "stress imprinting" as a primer induced by the first exposure to a stress that leads to enhanced resistance to a later stress 47,48 . ROS, as signaling molecules, play a key role in such priming events 49 . The harpin protein, Hpa1 Xoo , which is isolated from Xanthomonas oryzae, confers resistance to Verticillium wilt by activating a priming mechanism in cotton. A rapid burst of ROS was observed in Hpa1 Xoo -transformed plants after inoculation with V. dahliae 14 . Our previous study showed that Hcm1 protein possesses the same characteristics as the harpin protein Hap1 Xoc 22 . In addition, Hcm1, like Hpa1 Xoc , is located in the plasma membrane (Fig. 6a), which may be necessary for the function of harpins 20,42,43 . In Hcm1-transformed plants, H 2 O 2 content was slightly higher than in parent W0 plants and a ROS burst occurred after inoculation with V. dahliae (Fig. 4a,b). SA and ROS interplay in the transcriptional control of defense gene expression and play an important role in the disease resistance of plants 50 . On infection of hexanoic acid-treated plants, hexanoic acid activates the SA pathway as part of the priming mechanism 51 . Recent studies show that NO is another key signaling molecule involved in the induction of protection against biotic and abiotic factors through a complex network 40 . Harpins can activate the expression of PR genes such as NPR1, PR1-a (an SA marker) [21][22][23] , and HSR203J and HIN1 (HR markers) 21,41,48,52 . The up-regulation of GhNPR1, GhPR1, and GhNOA1 in response to pathogen infection was observed in Hcm1-transformed plants, revealing that the signaling pathways of SA and NO were activated (Fig. 4d). These results indicate that Hcm1 may lead to a primed state in cotton, and result in a faster and stronger induction of basal resistance mechanisms upon pathogenic attacks, since the priming was accompanied by an ROS burst, SA accumulation, and the induction of PR genes 14,53 . All harpins reported thus far, except XopA 54 and truncated HrpZ1 55 , can induce an HR in plants. Hcm1, which contains a harpin, also induces an HR in planta 14,22 . In Hcm1-transformed plants, the HR marker genes, HSR203J and HIN1, were activated (Fig. 4c) and a micro-HR was observed after inoculation with V. dahliae (Fig. 5). Miao et al. 14 suggested that such a micro-HR may augment the response to infections caused by fungal pathogens.

Hcm1 may provide antimicrobial properties in cotton. Cecropin A-melittin of Hcm1 protein has been
shown to effectively inhibit the growth of a variety of pathogens in vitro 22 . The Hcm1 protein extracted from prokaryotic expression or transgenic line H213 plants inhibited the growth of V. dahliae and F. oxysporum in vitro (Fig. 6b). The Hcm1 protein was located in the plasma membrane of plant cells (Fig. 6a), which may be help cecropin A-melittin to inhibit pathogens. In Hcm1-transformed plants, the biomass of V. dahliae was markedly lower than in parent W0 plants, as determined by qRT-PCR analysis and by observing the fluorescent signal strength of V. dahliae harboring the GFP gene (Fig. 6d). These results showed that the spread of V. dahliae was effectively hindered. A synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties, MsrA1, effectively restricts Alternaria brassicae and Sclerotinia sclerotiorum infection in transgenic Brassica juncea and Solanum tuberosum plants 34,56 . The novel cecropin A-melittin hybrid peptide, CEMA, which has strong antimicrobial activity in vitro, confers resistance against Fusarium solani in transgenic tobacco 57 . Our previous studies have shown that the disease resistance conferred by Hcm1 protein is more effective than that of the Hpa1 Xoc protein when Hcm1 or Hpa1 Xoc proteins are sprayed onto plants 22 . These results indicate that the cecropin A-melittin of the Hcm1 protein may also contribute to resistance against Fusarium and Verticillium wilts in transgenic cotton. The improved resistance to Fusarium and Verticillium wilts in cotton plants conferred by the Hcm1 protein may be a joint action of the Hpa1 Xoc protein and cecropin A-melittin.
In conclusion, these results lead us to suppose that the Hpa1 Xoc protein from Hcm1 activates a ROS priming mechanism in transgenic plants in response to V. dahliae and F. oxysporum infection, and cecropin A-melittin from Hcm1, which is located in the plasma membrane, simultaneously inhibits the spread of V. dahliae and F. oxysporum to confer resistance to both Verticillium and Fusarium wilts in cotton.

Future potential for fusion protein.
Verticillium wilts are among the most devastating fungal diseases worldwide and affect hundreds of different plant species including high value agricultural crops 58 . Economic losses of 50% or higher commonly occur in high value crops, including cotton 59 , lettuce 60 , olive 61 , and potato 62 . F. oxysporum was also described as an important fungal pathogen in a survey of plant pathologists, based on its economic and scientific importance 63 . Plant genetic engineering has been made possible thanks to extensive research conducted during the last three decades. Several studies have reported the control of F. oxysporum and V. dahliae infection by transgenic approaches [6][7][8][9][10][11][12][13][14] . Hcm1, a novel protein that induces plant defense responses and directly inhibits microbial growth, could improve cotton resistance to Verticillium and Fusarium wilts and offer a considerable yield advantage. Our previous 22 and present studies showed that Hcm1 confers resistance to multiple pathogens either by spraying on plants or expressing in plants, indicating that fusion proteins like Hcm1 could be widely applied to other crops in future to improve defense against plant diseases and improve crop yields.

Materials and Methods
Plant materials and V. dahliae and F. oxysporum culture. The transgenic cotton plants, as well as their parent W0, the resistant control for Verticillium wilt and Fusarium wilt, Gossypium barbadense (G. barbadense) cv. Hai7124, and the susceptible control for Verticillium and Fusarium wilts, G. hirsutum cv. Junmian 1, were grown in the green house facility in Nanjing Agricultural University in China. The growing conditions were: a constant temperature of 28 °C, a relative humidity of 70%, and a 16 hr photoperiod. Highly aggressive defoliating V. dahliae isolate V991 was stored in our laboratory and non-defoliating V. dahliae isolate BP2 was provided by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. V. dahliae V991 harboring the GFP gene was provided by the Biotechnology Institute, Jiangsu Academy of Agricultural Sciences. V. dahliae was maintained on PDA at 25 °C for 5 days, inoculated into Czapek's medium 64 , and then shocked at 25 °C for 5 days. F. oxysporum isolate Fnj1, which was provided by Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, was maintained on PDA at 24 °C for 3-5 days, inoculated into Czapek's medium and shocked at 25 °C for 3 days. Before inoculation, the conidia were counted and the conidia suspension was adjusted to the required density with distilled water.

Cotton transformation and transgenic plant selection. The recombinant binary vector
pBI35S-Hcm1-NPTII, which contained aneomycin phosphotransferase II (NPTII) with a nopaline synthase (Nos) promoter and terminator, a CaMV35S promoter, an Hcm1 insert, and a Nos terminator, was transformed into W0 plants. Agrobacterium-mediated cotton transformation was performed as described previously 65 . After induction, differentiation, and plantlet regeneration, the plantlets were grafted onto rootstocks and grown in a greenhouse. The homozygosity of transgenic plants were determined by analyzing the segregation ratio of the kanamycin selection marker and by PCR analysis. Kanamycin resistance tests, PCR analysis, Southern blots, Western blots, and resistance to Verticillium wilt were used to screen T 1 to T 6 progeny for Hcm1-transformed cotton lines.

Southern and Western blots analysis.
The method of Southern blots analysis was conducted as described by Lv et al. 66 . 20 μ g gDNA from the leaves of Hcm1-transformed and parent W0 plants was digested with EcoRI. Probes were prepared from purified PCR products of the NPTII coding region. The labeling of probes, prehybridization, hybridization, and detection were performed according to the protocol of the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science, Mannheim, Germany).
Total protein was extracted from the leaves of Hcm1-transformed and parent W0 plants according to the manufacturer's instructions of the Plant Protein Extraction Kit (CWBIO, Beijing, China). Protein concentration was measured according to the manufacturer's instructions for the BCA Protein Assay Kit (Solarbio, Beijing, China). Total proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Roche Applied Science, Mannheim, Germany). The membrane was blotted with a polyclonal antibody developed against Hcm1 and a goat anti-rabbit IgG-HRP antibody (Sino-American Biotech, Luoyang, China). The color was developed using DAB.
qRT-PCR. Total RNA from leaves, stems and roots of Hcm1-transformed and parent W0 plants was isolated using the CTAB method 67 , and 2 μ g of total RNA was used for reverse transcription. EF-1α (Table S1) from cotton was used as an internal control for normalization of the different cDNA samples. The PR genes in cotton are listed in Table S2. The primer sequences for PR genes are shown in Table S1. PCR was performed using the real-time PCR system (Bio-Rad) along with SYBR Green PCR Master Mix (Applied Biosystems). Each PCR was repeated three times, and the data were evaluated using the comparative cycle threshold method described by Livak and Schmittgen 68 .
Evaluation of resistance to Verticillium wilt and Fusarium wilt in greenhouse conditions. For the determination of Verticillium wilt resistance, after surface disinfection for 5 min with a 5% solution of sodium hypochlorite, cotton seeds were sown in a potting mixture (peat:vermiculite, 1:1, v/v). Thirty 18-day-old cotton seedlings were inoculated with defoliating V. dahliae isolate V991 and non-defoliating V. dahliae isolate BP2 by soil drenching with 20 ml conidial suspension (5 × 10 6 conidia/ml) for each pot (250 ml), and were grown under the following conditions: 12 hr of light at 25 °C and 70-90% relative humidity. Plants in the control group received same amount of sterile water. The DI was measured after two weeks in a greenhouse. After inoculated with non-defoliating V. dahliae isolate BP2, foliar damage was evaluated by rating the symptom on the cotyledon and leaf of inoculated plant according to the following disease grades: 0 = healthy plants, no fungal infection, 1 = 25% of the leaves showing yellowing or abnormal yellow spots, 2 = 25 to 50% of the leaves showing yellow spots, 3 = 50 to 75% of the leaves showing brown spots and curled leaf edges, and 4 = > 75% of the leaves showing yellow spots or irregular yellow spots between the main vein of leaves. After inoculated with defoliating V. dahliae isolate V991, foliar damage was evaluated by rating the symptom on the cotyledon and leaf of inoculated plant according to the following disease grades: 0 = healthy plant, 1 = yellowing or necrosis of 1-2 cotyledons, 2 = yellowing or necrosis of 1 true leaf, 3 = more than 2 wilted or necrotic leaves, 4 = no leaf left or dead plant.
For the determination of F. oxysporum resistance, a spore suspension of Fnj1 was added to sterilized strain bags containing grains of wheat. The grains of wheat were removed and dried after 20 days, before being mixed with mould and sand (1:1, v/v) at a ratio of 3% and encased an aluminum skin frame (45 cm × 33 cm × 16 cm). After surface disinfection, cotton seeds were sown in the aluminum skin frame and grown with 12 hr of light, at 25 °C and 70% -90% relative humidity. The DI was measured after seven weeks in a greenhouse. After inoculated with F. oxysporum isolate Fnj1, foliar damage was evaluated by rating the symptom on the cotyledon and leaf of inoculated plant according to the following disease grades: 0 = healthy plants, no fungal infection, 1 = 25% of the leaves showing yellowing or wilting, 2 = 25 to 50% of the leaves showing yellowing or wilting, leaf veins showing yellow, 3 = 50 to 75% of the leaves showing yellowing or wilting, cotton plants dwarf or wilting, and 4 = > 75% of the leaves showing yellowing or wilting, cotton plants dead.
The disease index was calculated as according to the formula: DI = [∑disease grades × number of infected plants)/(total checked plants × 4)] × 100 9,11 . At least thirty individual plants per line were subjected to resistant analysis and each experiment was repeated four times.
Evaluation of resistance to Verticillium and Fusarium wilts in field conditions. For resistance assessment, the transgenic lines, the parental line W0, and the resistant variety, Hai7124 were grown in the Verticillium wilt nurseries in the Henan and Xinjiang provinces in China. To assess their agronomic performance, the transgenic lines and the parental line W0 were also planted at a farm without diseased soil in Jiangsu Province, China, in 2014. All seeds were treated with an insecticide (Pymetrozine, J&K, San Diego, USA) prior to planting to protect against thrip and aphid damage, and some seeds were also treated with dynasty fungicide to control damping off diseases. In the Verticillium wilt nurseries of Henan province and Xinjiang province, the seed densities were 5 seeds m -1 and 25 seeds m -1 , respectively. Each treatment was replicated four times, and the replicates were arranged in a randomized complete block design. The DI was calculated using the above formula. At the completion of the trial, fifteen successive plants were chosen and tagged in each plot. The height of plants, number of fruit branches, and boll number per plant were investigated. Twenty-five bolls were manually harvested in each plot to investigate the weight of boll and lint percentage. Total cotton yield was assessed by hand picking all harvestable bolls. Subcellular localization of Hcm1. The full length Hcm1 coding region was inserted between the cauliflower mosaic virus 35S promoter and terminator sequences in the pJIT166-GFP vector by PCR with linker primers (Table S1) that contained HindIII and XbaI sites. All plasmid constructs were confirmed by sequencing. The 35S::GFP control and 35S::Hcm1::GFP vectors were transiently expressed in onion epidermal cells using a biolistic particle delivery system (PDS-1000 Bio-Rad, USA). The subcellular localization of the 35S::Hcm1::GFP fusion protein was observed with a confocal laser scanning microscope (LSM 510, Zeiss, Germany).

Observation of ROS burst and quantification of H 2 O 2 in cotton leaves.
The third cotton leaves that had no visible wounds were selected when the plants were at the 4 leaf stage and the leaf surfaces were smeared with the conidial suspension of V. dahliae (1-3 × 10 7 ml) and incubated for 0, 0.5, 1, 3, 5, 8, or 12 hr. To visualize the accumulation of H 2 O 2 , the fresh cotton leaves were collected and incubated in 1 mg/ml of DAB (pH 3.8) for 8 hr and then decolorized in 96% ethanol. The samples were examined with a stereo microscope lens (Olympus, DP72, Germany). The accumulation of H 2 O 2 was visible as red-brown discoloration. The production of H 2 O 2 in leaves was also measured with a commercial H 2 O 2 detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) using the method described by Jiang and Zhang 69 and expressed as a percentage of fresh weight. Leaves dipped in sterile water were used as the negative control. Each experiment was repeated six times.
Microscopic investigation of micro hypersensitive response. The third cotton leaves that had no visible wounds were selected and the leaf surfaces were smeared with the conidial suspension of V. dahliae (1-3 × 10 7 ml). Leaves were collected at 0, 0.5, 1, 3, 5, 8, or 12 hr and stained with trypan blue using the method described by Lipka et al. 41 . In addition, roots of transgenic line H213 and parent W0 plants at the 2 to 3 leaf stage were inoculated with a conidial suspension of V. dahliae (5 × 10 6 /ml) according to the method described above. Leaves were collected 15 days after inoculation with V. dahliae and then stained with trypan blue. Stained leaf samples were observed under a Leica light microscope (Leica DMRB, Leica Microsystems, Germany) and photographed with a Leica DFC camera (DM2500-3HF-FL, Leica Microsystems, Germany). Leaves without any wounds or visible symptoms of the disease from 10 independent H213 plants were examined.
Qualitative and quantitative detection of V. dahliae in cotton. Roots of transgenic line H213 and parent W0 plants at the 2 to 3 leaf stage were inoculated with a conidial suspension of V. dahliae harboring the GFP gene (5 × 10 6 /ml) according to the method described above. 15 days post-inoculation, the fluorescence signal strength at leaves and the rhizome connections were detected using a laser scanning confocal microscope (LSM 510, Zeiss, Germany). As the quantity of V. dahliae increases, the intensity of the fluorescent signal from GFP also increases. The roots, lower half of the stems, upper half of the stems, and first true leaves were also collected to use for DNA extraction. The internal transcribed spacer region of the ribosomal DNA was targeted to generate a 200 bp amplicon to measure the biomass of V. dahliae, using the fungus-specific primer W9500F 70 and the V. dahliae-specific reverse primer W9500R 71 . EF-1α from cotton was used as an internal control for normalization of the different DNA samples. The average fungal biomass was determined using at least six V. dahliae-inoculated plants for each genotype, and quantified in plants as described by Ellendorff Ursula et al. 72 .
Antimicrobial spectrum for Hcm1. CFEPs of Hcm1 were obtained using prokaryotic expression technology according to the methods described by Che et al. 22 . Hcm1-transformed protein was also extracted from the leaves of Hcm1-transformed plants. 10 μ l spore suspension of V. dahliae isolate V991 and F. oxysporum isolate Fnj1 was placed in the center of PDA or CM plates and then 5 mm diameter holes were made around the mycelial discs using a hole puncher. One hundred microliters of Hcm1 protein was added to the holes on the PDA or CM plates. The plates were incubated at 28 °C for 3-5 days depending on the fungal growth rate. Carbendazim (50 μ g ml −1 ) was used as the positive control for the fungi. The crude cell-free vector preparations (CFVPs) and the proteins from parent W0 plants were used as negative controls.