Response of AtNPR1-expressing cotton plants to Fusarium oxysporum f. sp. vasinfectum isolates

Abstract

In our earlier investigation, we had demonstrated that transgenic cotton plants expressing AtNPR1 showed significant tolerance to Fusarium oxysporum f. sp. vasinfectum, isolate 11 (Fov11) and several other pathogens. The current study was designed to further characterize the nature of the protection provided by AtNPR1 expression and its limitations. Green Fluorescent Protein-expressing Fov11 was generated and used to study the progression of the disease within the plant. The results show that the spread of the pathogen was slower in the AtNPR1-transformants compared to the wild type plants. Transcript analysis in the seedling root and hypocotyl showed that the transgenic lines are capable of launching a stronger defense response when infected with Fov11. We further confirmed that AtNPR1 transformants showed greater degree of tolerance to Fov11. However, little or no protection was observed against a related, but more virulent isolate, Fov43, and a highly virulent isolate, CA9.

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The online version of this article (doi:10.1007/s12298-016-0411-x) contains supplementary material, which is available to authorized users.

Introduction

Several organisms such as fungi, nematodes and bacteria can cause diseases in cotton resulting in significant yield losses (Hillocks 1992). The average annual cotton production loss due to diseases and nematodes in the United States and other cotton producing countries is approximately 12% (Blasingame and Patel 2001). Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), reduces cotton production by ~100,000 bales per year in the U.S. (Davis et al. 2006). There are six recognized races of Fov, cotton cultivars with resistance to all races do not exist and host resistance is the most effective approach for managing Fusarium wilt of cotton (Cianchetta and Davis 2015).

In plants, NPR1 (Non-expressor of Pathogenesis Related genes-1) is a key regulator of salicylic acid mediated systemic acquired resistance against pathogens (Cao et al. 1994; Shah et al. 1997). Transgenic expression of NPR1 gene in various plant species has been shown to confer tolerance to a broad spectrum of diseases. Makandar et al. (2006) showed that the expression of Arabidopsis NPR1 gene in susceptible wheat cultivar conferred resistance to Fusarium head blight (FHB) caused by F. graminearum. They reported that this resistance against FHB is associated with faster activation of defense response in AtNPR1-expressing transgenic wheat. In another study, Malnoy et al. (2007) overexpressed a homologous NPR1 (MpNPR1) in apple. They showed that the transgenic apple plants exhibited increased resistance against apple scab, cedar apple rust and fire blight diseases. Zhang et al. (2010) reported that over-expression of AtNPR1 in citrus increased resistance against citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. Citri (Xcc). A recent study showed enhanced resistance against Huanglongbing disease in citrus plants expressing the AtNPR1 gene (Dutt et al. 2015). In our earlier investigations, we found that expression of AtNPR1 in cotton conferred protection against two necrotrophic fungal pathogens, R. solani, (a cause of seedling damping-off) and A. alternata (a cause of foliar disease), two wilt diseases, Fusarium wilt caused by Fov and Verticillium wilt caused by nondefoliating pathotype of Verticillium dahliae, black root rot caused by T. basicola, and reniform nematodes (Parkhi et al. 2010a; Parkhi et al. 2010b; Kumar et al. 2013).

In the current investigation, we examined disease progression and defense response of AtNPR1 transformants at the molecular level, in response to a challenge with Fov11 isolate. In addition, we examined whether the AtNPR1-mediated protection observed earlier against Fov11, could be sustained with a related isolate, Fov43, and another highly virulent isolate, CA9.

Materials and methods

Plant material

Transgenic cotton plants (Coker 312) expressing an AtNPR1 gene under the control of CaMV 35S promoter have shown resistance to multiple fungal pathogens, including Fov11 (Parkhi et al. 2010a; Kumar et al. 2013). T4 generation plants of a transgenic line, 68L-19, were used in the current investigation.

Fusarium isolates

Two isolates belonging to VCG1a, namely Fov11 (ATCC 46644) and Fov43 (obtained from Insect Control & Cotton Disease Research Unit, Southern Plains Agricultural Research Center, USDA-ARS, College Station, TX) and CA-9 (race 4, a highly virulent isolate) were used to study the response of wild type (WT) and transgenic cotton plants.

Generation of GFP-expressing Fov11 and measurement of disease progression

sGFP expression vector pCT74 was used to transform fungal protoplasts (Lorang et al. 2001). The protoplast preparation and transformation were carried out as described (Hohn and Desjardins 1992; Oren et al. 2003) with minor modifications. A few blocks of Fov11 from potato dextrose agar culture were inoculated in 100 ml potato dextrose broth and grown for 10 days on a rotary shaker at 25 °C at 150 rpm. The culture was filtered through a sterile miracloth and centrifuged at room temperature at 5000 rpm for 10 min. The pellet was resuspended in 4–5 ml of the supernatant and a spore count of 10⁸ was mixed in 100 ml of YEPD broth containing 0.3% Yeast Extract (Difco laboratories, Detroit, MI), 1% Bacto™peptone (Difco Laboratories, Detroit, MI) and 2% Dextrose (d-glucose) broth and grown for 12–14 h at 25 °C at 175 rpm on a rotary shaker. Protoplasts were prepared and regenerated as described by Hohn and Desjardins (1992) with the exception that the fungal cell walls were digested with 500 mg driselase, 1 mg chitinase, and 100 mg lysing enzyme (Sigma Chemical Co., St. Louis) and the regeneration/transformant selection was performed in plates containing low melting temperature agarose (0.65%) in TB3 (0.3% yeast extract, 0.3% casamino acids and 20% sucrose) supplemented with 50 mg/L ampicillin (Sigma Chemical Co., St. Louis) and 50 mg/L hygromycin B (Sigma Chemical Co., St. Louis). GFP-expressing Fov11 transformants were selected with the aid of fluorescence microscope (Zeiss Stemi SV11 with GFP filters; excitation: 470 nm and emission: 500–525 nm). Transgenic lines were examined for their virulence and one line was selected to examine disease progression in WT and 68L-19 cotton transformants.

Stem puncture method of inoculation was used to follow the progression of the infection and specifically to measure the spread of Fov into the shoot. GFP-expressing Fov11 was grown on plates containing Difco™ potato dextrose agar (PDA) medium supplemented with 50 mg/L of tetracycline for 1 week. A section of agar with sporulating mycelia was cut from the PDA plate and transferred to 2 mL sterile distilled water and vortexed for 5 s to prepare a conidial suspension. This conidial suspension was filtered through a sterile paper towel to remove mycelial debris. Conidial count was obtained using a haemocytometer and the final count was adjusted to 10⁷ conidia/ml by diluting with sterile distilled water. A 23-gauge needle was used to inoculate 4-week-old cotton plants using the stem puncture method (Bugbee and Presley 1967; Bolek et al. 2005). Distilled water was used in place of the inoculum for mock treatment. Seven days post-inoculation, stem sections cultured on potato dextrose agar (PDA) medium. Internode sections from infected plants were surface sterilized in 10% bleach for 10 min followed by thorough rinsing (5 times) with sterile distilled water. The bark was removed using a sterile scalpel, the first, second, third and fourth internodes were split longitudinally into two halves. Sections from each internode were cultured separately on individual PDA plates and incubated at 25 °C for 4 days. The presence of Fov11 in an individual internode was confirmed by observing the GFP-expressing mycelia with the aid of a fluorescence microscope (Zeiss M2 BIO Fluorescence Combination Zoom Stereo/Compound microscope; excitation: 470 nm and emission: 525 nm). A schematic diagram showing the strategy used to conduct this assay is shown in Fig. S1.

Fusarium wilt assay

Plants were grown for 2 weeks in sand before inoculating with Fov cultures, root-dip inoculation method was followed as described in a previous report (Parkhi et al. 2010a). To check the defense related gene expression analysis samples were harvested 24 h post infection (hpi) of the roots with conidial inoculum of Fov11. Controls consisted of mock treatment of the roots with water. To study the performance of cotton plants against high and low virulent isolates, various parameters such as stem height, stem weight, leaf weight and leaf disease index were recorded 2 weeks post-inoculation as described in Parkhi et al. (2010a) where plants were survived.

Defense related gene expression analysis

Gene expression analysis of the defense response was performed by northern blot hybridization in case of root samples. Total RNA was isolated using spectrum plant total RNA kit (Cat # STRN50-1Kt; Sigma, St. Louis, MO, USA). There were three biological replications for each treatment and the three replications were pooled to make one sample. A 6 µg RNA sample was fractionated on a denaturing agarose gel and transferred to a nylon membrane (Cat #RPN303B, Amersham Pharmacia). Primers were designed based on cotton EST sequences available in the NCBI database (Table S1). These primers were used to amplify target gene sequences from the cDNA prepared from infected root RNA. PCR products were purified, labeled with P³²-dCTP, and used as probes in Northern hybridization as previously describe by Kumar et al. (2013). qRT-PCR analysis was performed on hypocotyl samples to study the expression of GhPR1, GhLOX1, Ghchitinase and GhGlucanse genes. Three independent biological replicates of seedling hypocotyls were harvested at 24 hpi. An aliquot of 0.8 µg total RNA was used to synthesize cDNA using Taqman reverse transcription kit (part #N808-0234; Applied Biosystems, USA). Gene specific primers were used to conduct qRT-PCR analysis are listed in Table S2. All the reactions were carried out with three technical replicates. Transcript levels of the defense genes were normalized to GhHistone3. Wild-type mock treatment (WT-Mock) was used as a standard for comparison of expression levels. The induction of defense related genes was calculated by using relative quantification method, i.e. 2^−∆∆Ct (Li et al. 2004).

Statistical analysis

In the experiments each treatment consisted of 10 replications unless otherwise stated. The data were analyzed through one-way ANOVA and are presented as mean ± standard error.

Results

Progression of disease in transgenic and WT plants

GFP-expressing Fov11 was used to inoculate 4-week-old plants using stem puncture method. The plants were at the three or four internode stage of growth at the time of termination of the experiment. Individual internodes from each plant were cultured separately. In addition, the leaf that had fully emerged from the distal end of the uppermost node (3rd or 4th above the cotyledonary node) was also removed and its petiole cultured on a PDA plate to ascertain whether the pathogen had spread to this most distal tissue. Having a GFP-tagged Fov11 culture allowed us to disregard other pathogens, if any that survived the surface sterilization process. The results, presented in Fig. 1, show that the disease had progressed in both sets of plants from the point of inoculation, however, the extent of its spread was lower in the transgenic line compare to WT. This is reflected by the number of distal internodes that remained free of the pathogen. The results from the cultured petiole segments (an asterisk on top of the bar indicates that the fungal infection had progressed to the distal most leaf) further support the notion that pathogen spread is inhibited in the transgenic plants.

Fig. 1.

The extent of disease progression in a wild-type (WT) and b AtNPR1-expressing transgenic plants, 7 days post-inoculation with Fov11 by the stem puncture method. The presence of Fov was confirmed by culturing the stem sections on PDA plates. Asterisk indicates that the distal leaf petiole was infected, meaning that the infection had progressed to the distal-most leaf

Analysis of defense-related gene expression in root and hypocotyl tissues

The expression of nine different defense related genes were examined in the root tissue by northern blot hybridization. Results presented in Fig. 2 show that at least four of these genes, pathogenesis related-1 (GhPR1), lipoxygenease (GhLOX1), chitinase (GhChitinase), and glucanse (GhGlucanase) are induced at a higher level in the transgenic roots compared to their WT counterparts at 24 hpi in response to Fov11. Results from qRT-PCR analysis for the hypocotyl tissues are shown in Fig. 3. With the exception of GhChitinase, other three genes do not show any induction in WT hypocotyls in response to infection. In contrast, substantially elevated expression of GhPR1, GhChitinase and GhGlucanase was observed in the transformants. These results indicate that the AtNPR1 transgenic line is able to launch a stronger defense response compared to their WT counterparts. In the absence of a biotic challenge, the AtNPR1 transformants do not show increased activity of the defense-related genes with the exception of PR1 that showed slightly elevated expression.

Fig. 2.

Analysis of defense-related gene expression in the roots of the wild-type (WT) and AtNPR1-expressing cotton plants, 24 h post-inoculation with Fov11 by the root dip method

Fig. 3.

Analysis of defense related gene expression in the hypocotyl tissue of the wild-type (WT) and AtNPR1-expressing cotton plants, 24 h post-inoculation with Fov11 by the root dip method. M = Mock, I = Infected. Data represent mean ± SE for three biological replicate samples

Performance of AtNPR1 transgenic lines cotton plants against different Fov isolates

Wilt assays were conducted using the root-dip inoculation method to determine the response of AtNPR1 transgenic plants to various Fov isolates. In the first experiment Fov11 and Fov43 were used to infect the AtNPR1 expressing and WT plants. Two weeks following the inoculation, plant health parameters were examined. Following inoculation with Fov11, the transgenic plants were relatively healthy and showed significantly lesser disease severity compared to their WT counterparts (Figs. 4, 5). Stem height, stem weight and leaf weight were 27, 66, and 80% higher, respectively, in the transformants compared to their WT counterparts. The leaf disease index for the 68L-19 line was 67% lower in comparison to the non-transgenic plants. In contrast, Fov43 was equally damaging to the WT and the AtNPR1 transformants (Figs. 4, 5).

Fig. 4.

Disease severity observed in experiment #1 in the wild-type (WT) and AtNPR1-expressing cotton plants, 2 weeks post-inoculation with Fov11 and Fov43 by the root dip method, under growth chamber conditions

Fig. 5.

Performance of wild-type (WT) and AtNPR1-expressing line 68L-19 against Fov11 and Fov43. Parameters a. stem height, b. stem weight, c. leaf weight and d. leaf disease index were recorded at 2 weeks following the root dip method of inoculation with the pathogen in experiment #1, conducted in a growth chamber. Data represent mean ± SE, *P < 0.05; n = 10

This experiment was repeated for the second time using the same conditions. As seen in the first experiment, the AtNPR1-expressing transgenic plants were relatively healthy and showed significantly lesser disease severity compared to their WT counterparts following inoculation with Fov11. However, little, if any, protection was observed in the transgenic plants against Fov43. With the exception of one parameter (stem height), no significant differences were detected between the transgenic and WT plants (Figs. 6, 7). In a separate experiment, CA-9 (race 4, a highly virulent) isolate was used to infect both the AtNPR1 and WT seedlings. Following the infection, both sets of plants were killed within a week.

Fig. 6.

Disease severity observed in experiment #2 in the wild-type (WT) and AtNPR1-expressing cotton plants, 2 weeks post-inoculation with Fov11 and Fov43 by the root dip method, under growth chamber conditions. a Fov11, b Fov43, and c mock treatment

Fig. 7.

Performance of wild-type (WT) and AtNPR1-expressing line 68L-19 against Fov11 and Fov43. Parameters, a stem height, b stem weight, c leaf weight, and d leaf disease index were recorded at 2 weeks following the root dip method of inoculation with the pathogen in experiment #2. Data represent mean ± SE, *P < 0.05; n = 10

Discussion

Constitutive expression of homologous and heterologous NPR1 gene in several plant species has been shown to confer a broad-spectrum resistance to a variety of pathogens. In our earlier investigations with AtNPR1 expressing cotton lines, we found that the transformants exhibited significant tolerance to V. dahliae (non-defoliating isolates TS2 and EZ2) Fov11, R. solani, T. basicola, A. alternata, and also reniform nematodes (Parkhi et al. 2010a; Kumar et al. 2013). In this study we examined the disease progression using GFP-expressing Fov11 culture, which provided us with a reliable assay to examine the spread of the disease. The results presented in this study show clearly that while the transgenic plants are not immune to the disease, its progression is substantially inhibited. We conducted Northern blot analysis on root tissues and qRT-PCR analysis on hypocotyl segments following inoculation of plants with Fov11. Results obtained in this study confirm that several defense-related genes are induced at higher levels in the AtNPR1 transformants compared to their non-transgenic counterparts. Similar results, showing higher-level induction of down-stream defense genes, have been obtained by others in wheat and apple (Makandar et al. 2006; Chen et al. 2012). It is interesting that the WT plants show little, if any, induction of these defense-related genes. Also, note that in the absence of a biotic challenge, the basal level activities of most of these genes are not elevated in the AtNPR1 transformants. Thus, in this regard we have further confirmed the results of our earlier studies (Parkhi et al. 2010a; Kumar et al. 2013).

Based on the promising results against a broad spectrum of pathogens (Parkhi et al. 2010a; Kumar et al. 2013), we wondered if the tolerance would extend to other Fov isolates and especially, those known to be more virulent compared to Fov11. In two comparative experiments in the current investigation, while significant tolerance was observed in the AtNPR1 transformants against Fov11, no such protection was seen against the more virulent isolate, Fov43. Next, we tested AtNPR1 transformants against a highly virulent isolate CA-9 that belongs to race 4. Both WT and transgenic plants died within a week following inoculation.

In conclusion, while AtNPR1 expression confers significant tolerance to the milder isolate Fov11, it is unable to provide any defense against the more virulent Fov isolates. Similar limitations were observed in the AtNPR1 transformants against Verticillium wilt, where protection was observed only against the non-defoliating isolates of V. dahliae and not the defoliating isolates (Parkhi et al. 2010b). Nevertheless, there are a large number of reports that show that NPR1 overexpression confers a strong protection against a variety of diseases (Lin et al. 2004; Makandar et al. 2006; Malnoy et al. 2007; Wally et al. 2009; Zhang et al. 2010; Dutt et al. 2015; Silva et al. 2015). Thus, controlled expression of NPR1 gene may still prove to be a useful means to confer tolerance to some pathogens in the field.

Electronic supplementary material

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