The heterologous expression of a Glycine max homolog of NONEXPRESSOR OF PR1 (NPR1) and α-hydroxynitrile glucosidase suppresses parasitism by the root pathogen Meloidogyne incognita in Gossypium hirsutum

ABSTRACT Experiments in Glycine max (soybean) identified the expression of the salicylic acid signaling and defense gene NONEXPRESSOR OF PR1 (NPR1) in root cells (i.e., syncytium) parasitized by the plant parasitic nematode Heterodera glycines undergoing the process of resistance. Gm-NPR1-2 overexpression in G. max effectively suppresses parasitism by H. glycines. The heterologous expression of Gm-NPR1-2 in Gossypium hirsutum impairs the ability of the parasitic nematode Meloidogyne incognita to form root galls, egg sacs, eggs and second-stage juvenile (J2) nematodes. In related experiments, a G. max β-glycosidase (Gm-βg-4) related to Lotus japonicus secreted defense gene α-hydroxynitrile glucosidase LjBGD7 suppresses M. incognita parasitism. The results identify a cumulative negative effect that the transgenes have on M. incognita parasitism and demonstrate that the G. max–H. glycines pathosystem is a useful tool to identify defense genes that function in other agriculturally relevant plant species to plant parasitic nematodes with different strategies of parasitism.


Introduction
Salicylic acid (SA) is an important hormone that is sensitive to increased environmental temperature, but functions effectively in plant defense to pathogens (Malamy et al. 1992;Cao et al. 1994). The genetic pathway for SA signaling has been determined in the plant genetic model Arabidopsis thaliana. In this genetic pathway, the lipase ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), which functions upstream of events directly involved in SA synthesis, heterodimerizes with the lipase PHYTOALOEXIN DEFICIENT4 (PAD4) (Zhou et al. 1998;Falk et al. 1999;Feys et al. 2001). SAL-ICYLIC-ACID-INDUCTION DEFICIENT2, a putative chloroplast-localized isochorismate synthase and its allelic ENHANCED DISEASE SUSCEPTIBILITY16 (EDS16) in conjunction with the multidrug and toxin extrusion efflux transporter EDS5, function downstream to activate SA biosynthesis. These interactions lead to the expression of NON-EXPRESSOR OF PR1 (NPR1) (Cao et al. 1994;Delaney et al. 1995;Glazebrook et al. 1996;Shah et al. 1997;Nawrath & Métraux 1999;Nawrath et al. 2000;Wildermuth et al. 2001). In NPR1-dependent SA signaling, SA binds to NPR1, stimulating its movement to the nucleus where it dimerizes with the TGA2 transcription factor in the presence of copper ions (Kinkema et al. 2000;Fan & Dong 2002). The complex then binds to the as-1 promoter sequence 5 ′ TGACGT3 ′ , driving target gene expression (Fan & Dong 2002). An identified downstream transcriptional target gene is the secreted protein PR-1 (Niggeweg et al. 2000). Ultimately, gene expression that results in a successful defense response occurs.
Microarray and RNAseq experiments have identified the expression of Glycine max homologs of EDS1 and NPR1 in RNA samples isolated from parasitized root cells called syncytia produced by its major pathogen, the parasitic nematode Heterodera glycines, undergoing the natural process of resistance (Klink, Hosseini et al. 2010;Klink, Overall et al. 2010;Matsye et al. 2011). The Gm-EDS1 transcript has been observed to represent 0.03675 of the RNAseq tags identified in parasitized cells undergoing the process of resistance at 9 days post infection (dpi) . In other works, both Gm-EDS1 and Gm-NPR1 have been observed to be expressed at statistically significant levels in syncytia undergoing the process of resistance at 3 and 6 dpi (Klink, Hosseini et al. 2010;Klink, Overall et al. 2010). To examine their potential role in defense, functional experiments have then been performed, demonstrating Gm-EDS1 and Gm-NPR1 function during resistance in G. max to H. glycines parasitism (Pant et al. 2014). These results have been reinforced further by related experiments showing the heterologous expression of A. thaliana TGA2, PAD4 and NPR1 in G. max results in defense to H. glycines and the root knot parasitic nematode Meloidogyne incognita Matthews et al. 2014). Reciprocal experiments examining whether the G. max SA signaling genes Gm-TGA2, PAD4 and NPR1 function to suppress nematode parasitism in other plant systems or whether Gm-TGA2 and Gm-PAD4 suppresses nematode parasitism in G. max were not examined Matthews et al. 2014).
The knowledge that A. thaliana NPR1 functions to drive the expression of the secreted protein gene PR1 indicates that a functional secretion system is important to the defense of plants to parasitic nematodes. Such a role for the plant secretion system has been determined in the G. max-H. glycines pathosystem with the identification of alpha-soluble N-ethylmaleimide-sensitive factor attachment protein (α-SNAP) composing part of the major H. glycines resistance locus rhg1 in G. max (Matsye et al. , 2012. These results were confirmed by the observation that overexpression of the α-SNAP interacting protein, syntaxin 31 (Gm-SYP38), known to function at the cis face of the Golgi apparatus, functions effectively to impair H. glycines parasitism (Banfield et al. 1995;Lupashin et al. 1997;Leyman et al. 1999;Collins et al. 2003;Peng & Gallwitz 2004;Bubeck et al. 2008;Pant et al. 2014;Pant, Krishnavajhala et al. 2015). In contrast, suppressed Gm-SYP38 expression in the H. glycines-resistant genotype G. max [Peking/PI 548402] results in the normally resistant genotype becoming susceptible to the parasite (Pant et al. 2014).
In the analysis presented here, roots that have been shown to express a G. max NPR1 homolog (Gm-NPR1-2) in Gossypium hirsutum (Pant, McNeece 2015) have been infected by M. incognita. The expression of Gm-NPR1-2 in G. hirsutum results in the suppression of the formation of galls, the production of egg sacs, the production of eggs and the development of J2 nematodes as compared to controls lacking the transgene. During the course of these experiments, a G. max β-glycosidase (Gm-βg-4) has been identified as being expressed in G. max roots overexpressing its Gm-NPR1-2 gene, indicating that it performs a defense function during parasitism by H. glycines. The heterologous expression of Gm-βg-4 in G. hirsutum roots is shown to suppress gall formation, egg sac production, egg production and the development of J2 nematodes as compared to controls lacking the transgene. The experiments show that genes functioning in defense in the G. max-H. glycines pathosystem function effectively to control M. incognita parasitism in G. hirsutum. Furthermore, there appears to be a cumulative negative effect on the different stages of the M. incognita life cycle which is a characteristic of β-glycosidases that function in defense in other plant-pathogen systems (Zagrobelny et al. 2008).

Selection of candidate genes
The selection of candidate genes to be expressed heterologously in G. hirsutum to affect the defense response has been aided by mining data from published gene expression experiments performed in G. max that resulted in its resistance to H. glycines (Klink, Hosseini et al. 2010;Klink, Overall et al. 2010;Matsye et al. 2011). These experiments prove that the method works to identify candidate genes that function in G. max defense to H. glycines parasitism Matthews et al. 2013Matthews et al. , 2014Pant et al. 2014, Pant, Krishnavajhala et al. 2015. In brief, G. max [Peking/PI 548402] and G. max [PI 88788] are infected with H. glycines [NL1-Rhg/HG-type 7/race 3] , resulting in a resistant reaction as proven histologically in unengineered roots which is the natural resistance response found in these G. max genotypes (Ross 1958;Endo 1965Endo , 1991Klink et al. 2007Klink et al. , 2009Klink et al. , 2011Klink, Hosseini et al. 2010;Klink, Overall et al. 2010). Roots are processed for histology and laser microdissection (LM), a procedure that has been used to collect syncytia undergoing the defense response (Klink et al. 2005(Klink et al. , 2007(Klink et al. , 2009Klink, Hosseini et al. 2010;Klink, Overall et al. 2010). These procedures allow for the isolation of mRNA that then is converted to probe for hybridization onto the Affymetrix® Soybean GeneChip® (Klink et al. 2007(Klink et al. , 2009Klink, Hosseini et al. 2010;Klink, Overall et al. 2010;Matsye et al. 2011). The hybridizations are run in triplicate (arrays 1-3) using a probe derived from RNA isolated from LM-collected syncytia obtained from three independent replicate experiments each run independently in the two different H. glycines-resistant genotypes (Klink et al. 2007(Klink et al. , 2009Klink, Hosseini et al. 2010;Klink, Overall et al. 2010). For the gene to be considered expressed at a given time point in the analysis presented here, probe signal is measurable above threshold on at least half of the arrays (three of six total arrays), combining both G. max [Peking/PI 548402] and G. max [PI 88788] (six total arrays), p < .05 (Klink et al. 2007(Klink et al. , 2009Klink, Hosseini et al. 2010;Klink, Overall et al. 2010). The original analysis procedure is performed as follows: the measurement for a particular probe set (gene) transcript on a single array is determined using the Bioconductor implementation of the standard Affymetrix®detection call methodology (DCM) (Klink et al. 2007(Klink et al. , 2009Klink, Hosseini et al. 2010;Klink, Overall et al. 2010). DCM consists of four steps, including (1) removal of saturated probes, (2) calculation of discrimination scores, (3) p-value calculation using Wilcoxon's rank test and (4) making the detection call (present ). Ultimately, the algorithm determines if the presence of a gene transcript is provably different from zero (P), uncertain or marginal (M), or not provably different from zero or absent (A) (Klink et al. 2007(Klink et al. , 2009Klink, Hosseini et al. 2010;Klink, Overall et al. 2010;Matsye et al. 2011).

RNA sequencing
These procedures have been described in Pant, Krishnavajhala et al. (2015). In brief, G. max RNA is extracted from roots using the UltraClean® Plant RNA Isolation Kit (Mo Bio Laboratories®, Inc.; Carlsbad, CA) and treated with DNase I to remove genomic DNA (Matsye et al. 2012;Pant et al. 2014). The RNAseq procedures (Illumina® HighSeq 2500® platform; Eurofins MWG Operon; Huntsville, AL) identify transcript (tag) counts and chromosomal coordinates of the G. max genome (Schmutz et al. 2010) along with the associated gene ontology annotations (Harris et al. 2004) and are outlined here. The qualities of raw reads are checked using the program FASTQC. The genome sequence and annotation of G. max (Schmutz et al. 2010) are obtained from Phytozome v9.0 (dated: 27 November 2011). The abundance of transcripts across all samples is measured and compared (Trapnell et al. 2012) and default setting of the programs used unless specified. Briefly, the raw reads for each sample are mapped on G. max genome using TopHat v2.0.6 (Trapnell et al. 2009), followed by Cufflinks v2.0.2 (Trapnall et al. 2010) to assemble the mapped reads into transcripts. The fragments per kilobase of exon per million fragments mapped (FPKM) values are calculated for all genes in all samples (Trapnell et al. 2010).

Gene constructs and genetic transformations
The cloning of Gm-NPR1-2 and Gm-βg-4 has been described (Pant et al. 2014). Overexpression studies are performed using the pRAP15 vector transformation system (Matsye et al. 2012;Pant et al. 2014;Pant, McNeece et al. 2015). The transgenic roots are produced by using Agrobacterium rhizogenes strain 15834 (Pant, McNeece et al. 2015). Due to the way A. rhizogenes transfers the DNA cassettes between the left and right borders of the plasmid to the root cell DNA, the subsequent growth and development of the genetically engineered cell into a transgenic root results in the production of a plant that is a genetic mosaic with the shoot being nontransgenic and the root transgenic. Therefore, each individual transgenic root system functions as an independent transformant line (Tepfer 1984;Matsye et al. 2012). The DNA backbone of the engineered pRAP15 contains nucleotide sequences containing both the eGFP and the gene of interest (GOI), each with their own promoter and terminator sequences (Pant, McNeece et al. 2015). Thus, roots expressing eGFP will also contain the GOI. The GOI amplicons are cloned from G. max [Williams 82/PI 518671] and ligated into the directional pENTR/D-TOPO® vector (Invitrogen®). The reaction contents are transformed into chemically competent E. coli strain One Shot TOP10® and selected on kanamycin (50 μg/ml) according to the protocol (Invitrogen®). Gene sequences are confirmed by matching it to the G. max [Williams 82/PI 518671] accession (Schmutz et al. 2010;Pant et al. 2014). Amplicons are ligated into the pRAP15 destination vector using LR Clonase® (Invitrogen®). The pRAP15 control and engineered vector are used to transform chemically competent A. rhizogenes (Hofgen & Willmitzer 1988;Haas et al. 1995) on tetracycline (5 μg/ml) according to Matsye et al. (2012). Genetic transformation experiments resulting in heterologous gene expression in G. hirsutum are performed according to Pant, McNeece et al. (2015). RNA is extracted from G. hirsutum roots using the UltraClean® Plant RNA Isolation Kit (Mo Bio Laboratories®, Inc.; Carlsbad, CA) and treated with DNase I according to the manufacturer's instructions (Invitrogen®). The cDNA is synthesized from RNA using the SuperScript First Strand Synthesis System for real time-polymerase chain reaction (RT-PCR) (Invitrogen®) with oligo d(T) as the primer (Invitrogen®) according to the manufacturer's instructions. Transformation of G. hirsutum is carried out by using the chemically competent A. rhizogenes (Pant, McNeece et al. 2015). Chemical selection of A. rhizogenes is carried out on LB-tetracycline (5 μg/ml) plates (Pant, McNeece et al. 2015). A PCR reaction using pRAP15 primers that amplify the 717 bp eGFP and the 690 bp A. rhizogenes root-inducing (Ri) plasmid (EU186381) VirG gene (VirG) confirms that A. rhizogenes contains both plasmids prior to G. hirsutum transformation (Supplemental Table 1). The presence of the engineered amplicons in pRAP15 is confirmed by PCR using primers for the respective genes and DNA sequencing (Pant et al. 2014).
A. rhizogenes-mediated transformation of G. hirsutism The procedure and plants generated in Pant, McNeece et al. (2015) for the non-axenic transformation of G. hirsutum has been used in the analysis presented here. Seeds of G. hirsutum are planted in pre-wetted sterilized sand, germinated and grown for 14 days at ambient greenhouse temperatures (∼26-29°C). The plants are cut at the hypocotyl with a freshly unwrapped, clean and sterile scalpel in a Petri plate containing a 10-ml pool of the pRAP15-transformed A. rhizogenes. The procedure ensures that infection by A. rhizogenes occurs at the exact moment the plant root is cut. The rootless plants (∼25 plants per beaker) are placed in 400 ml beakers containing A. rhizogenes cultured in Murashige and Skoog (MS) media (1962), including vitamins (Duchefa Biochemie, The Netherlands) and 3.0% sucrose, pH 5.7 (MS media). Only the bottom 0.5 cm of the hypocotyl end of the plantlet is submerged in the MS. No chemical selection is performed during the cocultivation. G. hirsutum undergoes vacuum infiltration for 30 min. The vacuum then is released slowly, allowing the A. rhizogenes suspension to infiltrate the tissue. Cocultivation is performed overnight in MS media in the 400-ml beaker on a rotary shaker at 28°C without chemical selection. After an overnight cocultivation, the cut ends of G. hirsutum are placed individually 2-4 cm deep into fresh, coarse, non-sterilized, vermiculite (Palmetto Vermiculite Co., Woodruff, SC) in 50-cell flats. The 50-cell flat containing the G. hirsutum plantlets are placed in a covered 32 quart Sterlite® Clearview Latch Box®. The plants then are covered and grown at a distance of 20 cm from standard fluorescent cool white 4100 K, 32-watt bulbs emitting 2800 lumens (Sylvania®, Danvers, MA) for 14 days at ambient lab temperatures (∼22°C). The plants are subsequently uncovered and transferred to the greenhouse. Genetically engineered roots are identified by carefully dislodging the plant and root ball from its pot and inspecting them for the expression of eGFP using the Dark Reader Spot Lamp (Clare Chemical Research, Dolores, CO). The remaining vermiculite is removed from these plants by washing the root ball in distilled, deionized water in a 1000-ml plastic beaker. The easily identified untransformed roots, evident by lacking fluorescence, are excised from the plants. The resulting chimeric plants are genetic mosaics (having transformed roots and untransformed aerial stocks). The chimeras are planted in a sterilized 50-50 mixture of a Freestone fine sandy loam (46.25% sand, 46.50% silt and 7.25% clay) and a sandy (93.00% sand, 5.75% silt and 1.25% clay) soil and allowed to recover for two weeks prior to experiments.

The infection of G. hirsutum by M. incognita
Procedures involving M. incognita infection of G. hirsutum are performed according to Diez et al. (2003). The M. incognita race 3 population originally growing at Humphrey County, Mississippi, on G. hirsutum has been isolated from egg masses. M. incognita are bulked up under ambient greenhouse conditions. M. incognita race 3 population is confirmed by the North Carolina differential host test (Myers 1990). M. incognita are extracted from greenhouse cultures by gravity screening and centrifugal flotation (sucrose sp gr = 1.13) (Jenkins 1964). M. incognita eggs and J2 are extracted from Lycopersicum esculentum (tomato) by a 4-min root immersion in 0.525% NaOCl (Hussey & Barker 1973). The solution is subsequently poured through a 75-μm pore sieve nested over a 28-μmpore sieve. The collected eggs, present on the 28-μm-pore sieve, are placed in water maintained at 28 ± 1°C for 3 days. The J2s hatch and are collected in a 28-μm-pore sieve at 24-h interval over a period of 3 days. M. incognita J2s are maintained in water at 4 ± 1°C until inoculation (Tang et al. 1994). The quantity of inoculum is calculated in a Petri dish with an enumeration grid using a stereomicroscope. At the end of the experiment, M. incognita are extracted from the soil as described previously. M. incognita populations are determined, calculating number of galls, egg masses, eggs and J2. Further determination of life cycle stages is accomplished using a modified acid-fuchsin staining-destaining procedure (Byrd et al. 1983). M. incognita life-stage development has been described using a modified Christie's method (Christie 1946;Christie & Cobb 1946;Tang et al. 1994). Parasitism has been assayed at four different points in their life cycle by calculating gall number, egg mass number, egg number and number of juveniles.

Quantitative PCR
Primers used in quantitative polymerase chain reaction (qPCR) gene expression experiments are provided in Supplemental Table 1. The G. hirsutum S21 primers have been designed from Gossypium raimondii S21 (Gorai.009G233700.1) and serve as a control (Pant, McNeece et al. 2015). The qPCR experiments use Taqman® 6-carboxyfluorescein (6-FAM) probes and Black Hole Quencher (BHQ1) (MWG Operon; Birmingham, AL). The qPCR differential expression tests are performed using RNA samples isolated from three independent replicates. The qPCR reaction conditions include a 20-μl Taqman Gene Expression Master Mix (Applied Biosystems, Foster City, CA), 0.9 µl of 100 μM forward primer, 0.9 µl of 100 μM reverse primer, 2 µl of 2.5 µM 6-FAM (MWG Operon®) probe and 9.0 µl of template DNA. The qPCR reactions are performed on an ABI 7300 (Applied Biosystems®). The qPCR conditions include a preincubation of 50°C for 2 min, followed by 95°C for 10 min. This step is followed by alternating 95°C for 15 s followed by 60°C for 1 min for 40 cycles. The statistical analysis using 2 −ΔΔC t to calculate fold change is followed according to the derived formula presented in Livak and Schmittgen (2001).

Microscopy
Stereoscope images are obtained on a Wild Heerbrugg stereoscope. The lenses are Wild Heerbrugg Makrozoom 1:5 having a 6.3-32× scale. Image capture is carried out using the IMT i-solution computer package (IMT i-Solution Inc., Ho Chi Minh City, Vietnam).

Heterologously expressed Gm-NPR1-2 activates endogenous defense gene transcription
Gm-NPR1-2 and its upstream activator Gm-EDS1-2 are expressed in G. max roots undergoing the process of resistance (Supplemental Tables 2 and 3). G. max roots overexpressing Gm-NPR1-2 results in resistance to H. glycines parasitism, indicating that it could be expressed heterologously in G. hirsutum to affect the defense response to M. incognita (Pant et al. 2014). A candidate gene screen examining the expression of G. max defense genes occurring during its resistance to H. glycines has led to the identification of a 525 amino acid (aa) signal peptide-containing G. max β-glycosidase (Gm-βg-4 [Glyma11g13810]), which is also expressed in G. max roots undergoing the process of resistance (Supplemental Tables 2 and 3). The Gm-βg-4 gene is induced by 1.59-fold in G. max roots genetically engineered to overexpress NPR1-2 (Table 1). The demonstrated roles for β-glucosidases in plant defense have led to the experiments presented here in G. hirsutum (reviewed in Gleadow & Moller, 2014). The expression of NPR1-2 in Gm-NPR1-2-OE roots was presented in Pant et al. (2014).

Confirming heterologous expression of analyzed transgenes in G. hirsutum
Prior gene expression experiments demonstrate that the heterologous expression of Gm-NPR1-2 in G. hirsutum can be achieved (Pant, McNeece et al. 2015). Those G. hirsutum roots heterologously expressing Gm-NPR1-2 are compared here with roots heterologously expressing Gm-βg-4 and control roots lacking the expression of either transgene (Figure 1). While the experimental procedure presented here in G. hirsutum is complicated by the heterologously expressed G. max gene not being present in the control roots, experiments are presented here confirming the heterologous expression of Gm-βg-4 in G. hirsutum ( Figure 2). G. hirsutum plants heterologously expressing Gm-NPR1-2 or Gm-βg-4 have then been examined to determine their effect on M. incognita parasitism, analyzing gall formation, egg mass number, egg count and number of juveniles ( Figure 2).

Analysis of the effect of heterologous expression of Gm-NPR1-2 on M. incognita parasitism
An examination of the effect of the heterologous expression of Gm-NPR1-2 in G. hirsutum on M. incognita parasitism has been performed ( Figure 3). The impact that Gm-NPR1-2 expression has on M. incognita parasitism is presented in  two different ways. Firstly, these data are presented in relation to M. incognita parasitism in the whole root mass. However, to account for potential variations caused by differences in the mass of the root, analyses on gall formation, egg mass number, egg count and number of J2s have also been calculated per gram of root tissue (Figure 4, Supplemental Table 4). The percent decrease in gall formation, egg mass number, egg count and number of J2s for each of the three replicates is presented (Table 2). It is noted that there appears to be an accumulative negative effect occurring during the course of the M. incognita life cycle as revealed in the low levels of J2s (Table 3).

Analysis of the effect of heterologous expression of Gm-βg-4 on M. incognita parasitism
An examination of the effect of Gm-βg-4 expression in G. hirsutum on M. incognita parasitism has been performed ( Figure 5). The analysis examines its effect on gall formation, egg mass number, egg count and number of J2s in relation to the whole root mass and per gram of root tissue (Figure 6; Supplemental Table 5). The percent decrease for each of the three replicates is presented (Table 3). It is noted that there appears to be an accumulative negative effect occurring during the course of the M. incognita life cycle as revealed in the low levels of J2s (Table 3).

Discussion
A number of plant pathogenic nematodes that infect G. max are capable of parasitizing other plant species. Among them is M. incognita which is able to successfully parasitize G. hirsutum. The M. incognita life cycle is composed of six stages including the egg that is encased within an egg mass, four juvenile stages and the adult. During infection, the M. incognita J2s burrow into roots and establish a nurse cell called a giant cell from which they feed. However, unlike syncytia formed by plant parasitic nematodes such as H. glycines, the growth and development of the giant cell results in the formation of swollen root regions around the giant cells leading to the formation of galls. This characteristic of infection by M. incognita presents advantages for studying their pathology since the parasitized root regions are easily identifiable without the aid of histological procedures. M. incognita presents other advantages for the study of resistance to root parasitic nematodes. For example, plants parasitized by M. incognita allow for an analysis of the effect that the expression of gene cassettes have at different stages of the nematode life cycle and their influence on giant cell development through the examination of gall formation. Therefore, any cumulative negative effect exerted by the plant on nematode fitness can be determined since multiple stages in its life cycle can be easily quantified. In the analysis described here, results are presented showing the effect that the heterologous expression of Gm-NPR1-2 or Gmβg-4 has on four sequential processes associated with M. incognita development including gall formation, egg mass formation, egg production and J2s that have hatched.
Heterologously expressed G. max NPR1 in G. hirsutum effectively impairs M. incognita fitness The effective defense response that has been obtained in G. hirsutum by the heterologous expression of Gm-NPR1-2 is consistent with the observations, showing that its expression is important to the defense response of G. max to H. glycines (Pant et al. 2014). These results are consistent with observations showing that the heterologous expression of the A. thaliana SA signaling genes PAD4, TGA2 and NPR1 suppresses M. incognita development in G. max. Therefore, the results presented here confirm the importance of the SA signaling pathway in defense to parasitic nematodes Pant et al. 2014;Matthews et al. 2014).

Gm-β-glucosidase is part of a conserved cellular process that functions in defense
The results presented here show the heterologous expression of Gm-βg-4, having characteristics of a secreted protein, functions effectively in G. hirsutum to suppress parasitism by M. incognita. The result confirms prior observations of the importance of the plant secretion system playing a major role in the defense of plants to parasitic nematodes in general (Matsye et al. , 2012Pant et al. 2014, Pant, Krishnavajhala et al. 2015. For example, earlier observations have been made in G. max in an examination of its syntaxin 31 homolog Gm-SYP38 (Pant et al. 2014) and earlier analyses of the alpha-soluble N-ethylmaleimide-sensitive fusion proteinassociated protein (α-SNAP) (Matsye et al. , 2012. The results indicate that the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) will  Table 4. perform a prominent role in defense (reviewed in Jahn and Fasshauer 2012). Bioinformatics analyses show that Gm-βg-4 is most closely related to the root-specific Lotus japonicus α-hydroxynitrile glucosidase LjBGD7, belonging to a small family of enzymes involved in the production of cyanogenic α-hydroxynitrile glucosides (Morant et al. 2008;Takos et al. 2010). In the α-hydroxynitrile glucoside metabolic pathway, active α-hydroxynitrile glucosides are produced through a pathway involving cytochrome p450 79 D4 (CYP79D4) which converts an amino acid to an oxime. A second enzyme, CYP71, converts the oxime to an α-hydroxynitrile. The αhydroxynitrile is then converted to a cyanogenic monoglucoside by the activity of UDP-glucosyltransferase and can be stored in this form. Subsequent activity by α-hydroxynitrile glucosidase removes the glucose moiety, activating the α-hydroxynitrile. The α-hydroxynitrile is then metabolized by α-hydroxynitrile lyase or experiences a spontaneous event, resulting in the production of hydrogen cyanide (HCN) which is toxic to the pathogen. HCN is later detoxified by β-cyanoalanine synthase (Gleadow & Moller 2014). The effective and conserved function that Gm-βg-4 has on suppressing M. incognita parasitism indicates that the cognate α-hydroxynitrile glucoside is also present in G. hirsutum. While the G. hirsutum genome is not available at this point, comparative analysis of the G. max and G. raimondii genomes has identified closely related homologs of all components of the α-hydroxynitrile glucoside metabolism. Thus, it is evident that Gm-βg-4 is involved in synthesizing active α-hydroxynitrile glucosides in G. hirsutum. If so, our result is similar to those presented by Forslund et al. (2004), demonstrating that the heterologous expression of a Manihot esculenta (cassava) CYP79D2 in L. japonicus results in the accumulation of the cyanogenic α-hydroxynitrile glucosides lotaustralin and linamarin.
Gm-NPR1-2 and Gm-βg-4 have a cumulative negative impact on M. incognita parasitism In the analysis presented here, the percent effect that the heterologous expression of the Gm-NPR1-2 and Gm-βg-4 transgenes has on M. incognita parasitism in G. hirsutum increases at subsequent stages of its parasitism. This observation indicates a cumulative negative effect caused by expression of these transgenes. This observation is very similar to those observed by Zagrobelny, Bak, Ekstrøm et al. (2007), Zagrobelny, Bak, Olsen et al (2007), and Zagrobelny et al (2008), showing the cumulative negative effect of β-glucosidases at different and subsequent stages of the life cycle in the Lepidopteran Zygaena filipendulae grown on L. japonicus. Thus, it appears that the heterologous expression of Gm-NPR1-2 and Gm-βg-4 functions in a similar manner producing an effective action that progressively and negatively impacts the M. incognita parasitism in G. hirsutum. Since the genes function effectively against H. glycines parasitism in G. max, it appears that they would have a broad spectrum action.  White arrowheads point toward the developing egg masses. Bar = 1 cm. Statistical analysis of the effect of Gm-βg-4 expression in G. hirsutum is presented in Figure 6.

The use of chimeric plants in analyzing plant parasitic nematodes
The hairy root system used here has been employed in G. hirsutum to analyze plant parasitic nematode fitness in transgenic roots (Triplett et al. 2008;Pant, McNeece et al. 2015). Many plant parasitic nematodes including H. glycines and M. incognita, among others, are obligate root parasites. The infection of transgenic hairy roots of G. max and G. hirsutum by H. glycines and M. incognita, respectively, indicate that these organs have the identity of roots. This characteristic is clear when examining pathogenicity, revealing levels of parasitism in genetically engineered control plants lacking the transgene that are similar to unengineered control plants. Furthermore, histological observation demonstrates that the development of H. glycinesinduced syncytia or M. incognita-induced giant cells is similar in genetically engineered control plants lacking the transgene and unengineered control plants. These observations are consistent with the presence of root hairs and other morphological and anatomical features of roots in the hairy root organs, indicating that these structures are roots and function as roots (Tepfer et al. 1984). The plants produced by this experimental procedure, however, are genetic chimeras that have transgenic roots and nontransgenic shoots (Collier et al. 2005;Klink et al. 2009;Matsye et al. 2012;Pant, McNeece et al. 2015). The hairy root method is rapid, allowing for the production of libraries of genes that can rapidly and inexpensively be tested . Therefore, the method allows for the testing of genes in plants amenable to the procedure that are not tractable genetically. Genes that function effectively in defense then can be targeted in genetic screens or engineered in through homologous recombination or other screening procedures (Jinek et al. 2012).  Table 5.