The heterologous expression of conserved Glycine max (soybean) mitogen activated protein kinase 3 (MAPK3) paralogs suppresses Meloidogyne incognita parasitism in Gossypium hirsutum (upland cotton)

Two conserved Glycine max (soybean) mitogen activated protein kinase 3 (MAPK3) paralogs function in defense to the parasitic soybean cyst nematode Heterodera glycines. Gene Ontology analyses of RNA seq data obtained from MAPK3-1-overexpressing (OE) and MAPK3-2-OE roots compared to their control, as well as MAPK3-1-RNA interference (RNAi) and MAPK3-2-RNAi compared to their control, hierarchically orders the induced and suppressed genes, strengthening the hypothesis that their heterologous expression in Gossypium hirsutum (upland cotton) would impair parasitism by the root knot nematode (RKN) Meloidogyne incognita. MAPK3-1 expression (E) in G. hirsutum suppresses the production of M. incognita root galls, egg masses, and second stage juveniles (J2s) by 80.32%, 82.37%, and 88.21%, respectfully. Unexpectedly, egg number increases by 28.99% but J2s are inviable. MAPK3-2-E effects are identical, statistically. MAPK3-1-E and MAPK3-2-E decreases root mass 1.49-fold and 1.55-fold, respectively, as compared to the pRAP15-ccdB-E control. The reproductive factor (RF) of M. incognita for G. hirsutum roots expressing MAPK3-1-E or MAPK3-2-E decreases 60.39% and 50.46%, respectively, compared to controls. The results are consistent with upstream pathogen activated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI) functioning in defense to H. glycines. The experiments showcase the feasibility of employing MAPK3, through heterologous expression, to combat M. incognita parasitism, possibly overcoming impediments otherwise making G. hirsutum’s defense platform deficient. MAPK homologs are identified in other important crop species for future functional analyses. Supplementary Information The online version contains supplementary material available at 10.1007/s11248-022-00312-y.

The analysis presented here examines the effect that the heterologous expression of the G. max MAPK3-1 and MAPK3-2 has on M. incognita parasitism of G. hirsutum, providing key information on an important defense signaling node. The expression leads to a significant decrease in M. incognita parasitism. The results are placed into context by providing a relationship of these results to previously reported observations.

Bioinformatics
The A. thaliana proteome is used to obtain its 20 MAPK protein sequences, including MAPK3 (AT3G45640) (Arabidopsis Genome Initiative 2000). The A. thaliana MAPK protein sequences are used to extract the G. max MAPK3-1 and MAPK3-2 (MAPK3-1 and MAPK3-2) protein sequences from its housed proteome at from Phytozome (https:// phyto zome. jgi. doe. gov) through a Basic Local Alignment Search Tool program (BLAST) query (Altschul et al. 1990). The default settings, include Target type: Proteome; Program: BLASTP-protein query to protein database; Expect (e) threshold: -1; Comparison matrix: BLOcks SUbstitution Matrix 62 (BLO-SUM62); Word (W) length: default = 3; number of alignments to show: 100 allowing for gaps and filter query, in order that they appear on the BLAST program. (Goodstein et al. 2012;McNeece et al. 2019). The MAPK3-1 and MAPK3-2 protein sequences are used in pairwise comparisons employing the EMBOSS Program Needle, Version 6.6.0 to compare MAPK3-1 and MAPK3-2 in the Matrix; EBLO-SUM62; Gap open, 10.0; Gap extend, 0.5; End Gap Penalty, false; End Gap Open Penalty, 10.0; End Gap Extension Penalty, 0.5 (Henikoff and Henikoff, 1992). The MAPK3-1 and MAPK3-2 protein sequences are used in BLAST queries of the G. hirsutum proteome housed at Phytozome using the EMBOSS 001 EBLOSUM62 Matrix in default settings with a gap penalty of 10.0 and extend penalty of 0.5 to obtain their homologs (Henikoff and Henikoff, 1992;Goodstein et al. 2012). The conserved domain analyses for the MAPK3-1 and MAPK3-2 proteins are performed according to Lu et al. (2020) using Conserved Domain Database (CDD) v3.19 in default settings. Multiple protein sequence alignments are performed using Clustal Omega under default settings (Sievers and Higgins, 2014).

Plasmid details
The Gateway-compatible, 14,758 bp, pRAP15 plasmid expression (E) vector is used in the transgenic analysis of G. hirsutum (Matsye et al. 2012;Pant et al. 2015Pant et al. , 2016McNeece et al. 2017;Niraula et al. 2020a;Klink et al. 2021a). A related plasmid, pRAP17 (15,596 bp, based off of the p*7GWIWG2(II) backbone) designed for RNA interference (RNAi) work but not used in the analysis has been generated (Karimi et al. 2002(Karimi et al. , 2007Curtis and Grossniklaus 2003;Klink et al. 2009Klink et al. , 2021a. The pRAP15 plasmid is based off of the p*7WG2D vector, allowing the efficient directional cloning of genes at its attR recombination sites (Karimi et al. 2002(Karimi et al. , 2007Curtis and Grossniklaus 2003;Matsye et al. 2012;Klink et al. 2021a). The pRAP15 plasmid, having the chloramphenicol-ccdB (Cm[r]-ccdB) gene (Invitrogen) (ccdB) that is lethal to E. coli TOP10 cells and acts as a selectable marker at the site where the candidate resistance gene (CRG) (i.e., MAPK3-1 or MAPK3-2) would be engineered, also functions as the transgenesis control (pRAP15-ccdB) in gene expression, M. incognita parasitism and root mass experiments (Tam and Kline 1989;Bernard et al. 1991;Salmon et al. 1994;Karimi et al. 2002;Curtis and Grossniklaus 2003;Matsye et al. 2012;Klink et al. 2021a). Maintenance of the original, un-engineered, pRAP15 plasmid (lacking the insertion of a CRG transgene) is accomplished by the presence of the Cm(r)-ccdB lethality gene, selected using One Shot ccdB Survival 2 T1 R Competent Cells (Invitrogen) (Klink et al. 2021a). The tetracycline resistance gene (TetR), inserted outside of the left and right border, has been added during the development of pRAP15 to facilitate selection in E. coli or, importantly, A. rhizogenes or other bacteria. The MAPK3-1 and MAPK3-2 expression in G. hirsutum is driven by the figwort mosaic virus (FMV) sub-genomic transcript (Sgt) promoter consisting of a 301-bp FMV Sgt promoter fragment (sequence − 270 to + 31 from the transcription start site [TSS]) in pRAP15 (Bhattacharyya et al. 2002). The pRAP15 plasmid has been used to directionally clone the G. max MAPK3-1 and MAPK3-2 genes for overexpression in soybean but has been used here to obtain its heterologous expression in G. hirsutum ). The pRAP15 plasmid has 2 selectable reporters. The pRAP15 plasmid has the enhanced green fluorescent protein (eGFP) gene reporter for visual selection of transgenic plant tissue (Matsye et al. 2012;Klink et al. 2021a). The transcription of the eGFP gene is driven by the rolD promoter (Haseloff et al. 1997). The eGFP gene cassette is terminated by t35S translational terminator for effective visual reporting in plant tissue (White et al. 1985;Elmayan and Tepfer 1995;Haseloff et al. 1997). Furthermore, the pRAP15 plasmid has the Basta® selectable marker encoded by the bar gene which confers resistance to the herbicide bialphos, useful for tissue culture (Thompson et al. 1987;Rathore et al. 1993;Karimi et al. 2002Karimi et al. , 2007. The bar gene is driven by the nopaline synthase promoter and terminated by the nopaline synthase terminator (Klink et al. 2021a).

Genetic transformation of Agrobacterium rhizogenes
The pRAP15-MAPK3-1 and -MAPK3-2-containing plasmids are genetically transformed into Agrobacterium rhizogenes strain 15,834 (15,834) using the freeze-thaw method (Hofgen and Willmitzer 1988;Pant et al. 2015). During the 15,834 genetic transformation procedure, 250 μl of bacteria previously snap frozen and stored at -80 °C in a 1:1 v/v cells in LB:30% sterile glycerol solution is thawed on ice. Plasmid DNA (0.1-1 μg) is added to 15,834 bacterial cells and gently mixed. The mixture of 15,834 cells and plasmid DNA is incubated on ice for 5 min. The contents are then subsequently transferred to liquid N 2 for 5 min. The mixture is transferred to a 37 °C water bath for a period of 5 min. The reaction contents are then transferred to a culture tube containing 1 ml of LB medium with no antibiotics to allow for a recovery period for the bacteria as their TetR gene activity engages, placed in a shaking incubator at 28 °C, and incubated for 2 h. The 15,834 cells are then collected, centrifuged for 2 min at 5000 rpm. This step is followed by resuspension of the pelleted cells in 200 μl of LB medium, followed by the resuspended bacteria being spread on LB agar plates containing 5 μg/ml Tet for chemical selection at 28 °C (Pant et al. 2015). After 2 days the 15,834 colonies that underwent genetic transformation are picked to undergo a procedure that determines the presence of the eGFP gene, root inducing (Ri) plasmid, and the MAPK3-1 or MAPK3-2 gene by PCR using the appropriate primers (Hodges et al. 2004;Haseloff et al. 1997;Pant et al. 2016;McNeece et al. 2019) (Supplemental Table 1). The 15,834 colonies harboring the appropriate plasmids are then grown in 250 ml of LB medium containing 5 μg/ml Tet at 28 °C in a shaking incubator at 250 rpm for 14 h. Upon confirmation of adequate 15,834 growth at an OD 600 of 0.6-0.8, the culture is used for G. hirsutum transformation after a centrifugation and resuspension in Murashige and Skoog (MS) media including vitamins (Duchefa, catalog number M0222), pH 5.7 at ambient room temperature (~ 18-21 °C) (Murashige and Skoog 1962). Please see the next section for details.
Genetic transformation of G. hirsutum M. incognita-susceptible G. hirsutum (Phytogen 565 WRF) seeds are planted in pre-wetted sterilized sand for germination. Seedlings are grown for 14 days at ambient greenhouse temperatures (~ 26-29 °C), then removed from the sand and washed in sterile, deionized water. The roots are excised with a sterile razor blade, producing root-less G. hirsutum. Genetic transformation of G. hirsutum is done as described by McNeece et al. (2017). An overnight culture of 15,834 containing the desired plasmid is grown in YEB Agrobacterium Growth Medium (Bioworld), supplemented with 5 μg/ml Tet at 28 °C

Selection of transgenic G. hirsutum
The pRAP15 vector, containing the eGFP visual reporter gene, is used to accomplish the expression of a targeted CRG (Jefferson et al. 1987;Collier et al. 2005;Matsye et al. 2012;McNeece et al. 2017;Klink et al. 2021a). In control and experimental plants, the eGFP (driven by the rolD promoter), ccdB control gene (driven by the FMV-Sgt promoter), MAPK3-1 (driven by the FMV-Sgt promoter), and MAPK3-2 (driven by the FMV-Sgt promoter) genes that are engineered into the pRAP15 plasmid each have their own promoter and terminator sequences (Collier et al. 2005;Matsye et al. 2012;Pant et al. 2015Pant et al. , 2016McNeece et al. 2017;Niraula et al. 2020a;Klink et al. 2021a). Due to the manner that 15,834 transfers the DNA cassettes located between the left and right borders of the destination vector into the root cell chromosomal DNA, the subsequent growth and development of the stably transformed genetically engineered cell into transgenic roots results in the production of a plant that is a genetic mosaic called a composite plant (Tepfer 1984;Collier et al. 2005). The composite, genetically mosaic, plant has a transgenic root system and a non-transgenic shoot.
PCR DNA primer sequences are provided (Supplemental Table 1). Isolated RNA (according to Invitrogen) is used to produce cDNA from transgenic G. hirsutum root RNA. Confirmation of eGFP expression is performed by PCR according to Niraula et al. (2020a). PCR using cDNA produced from mRNA isolated from the pRAP15-ccdB control and the MAPK3-1 and MAPK3-2-expressing roots are used to demonstrate that MAPK3-1 and MAPK3-2 are expressed in the transgenic roots of G. hirsutum.

Infection by M. incognita
The M. incognita (race 3) are confirmed by the North Carolina differential host test and increased on Lycopersicum esculentum (tomato) under ambient greenhouse conditions (Jenkins, 1964;Hussey and Barker 1973;Myers 1990;Tang et al. 1994;Diez et al. 2003). Eggs are extracted from roots by placing the root system in a 0.625% NaOCl solution and agitating the roots for 4 min using a rotary shaker at 120 rpm. Eggs are rinsed with tap water, collected on a 25-μm-pore sieve, then processed by sucrose centrifugation-flotation at 240 g for 1 min (Jenkins 1964).
M. incognita eggs are placed in a modified Baermann funnel (Peraza-Padilla et al. 2013) on a slide warmer (Model 77) (Marshall Scientific, Brentwood, NH) and incubated at 31 °C for 5 to 7 days to obtain second stage juveniles (J2s) (Xiang et al. 2016). The J2s are collected on a 25-μm-pore sieve, transferred to 1.5 ml microcentrifuge tubes, centrifuged at 5000 g for 1 min, rinsed with sterile distilled water and centrifuged at 5000 g for 1 min. The J2 suspension is adjusted to 30 to 40 J2s per 10 μl of water (Xiang et al. 2016). M. incognita extraction is performed by gravity screening and centrifugal flotation (sucrose specific gravity = 1.13) (Jenkins 1964). M. incognita eggs and J2s are extracted from L. esculentum roots by a 4-min root immersion in 0.525% NaOCl (Hussey and Barker 1973). The hatched M. incognita J2s are maintained at 4 ± 1 °C in water until inoculation (Tang et al. 1993). Transgenic G. hirsutum plants are grown in 15 cm diameter clay pots. The pots are filled with 500 cm 3 of the sterilized soil mixture that is 80% sand, 10% clay, and 10% silt. In these pots, a suspension of 2500 M. incognita J2s in 3 ml suspension are pipetted, divided into 2, 1.5 ml aliquots, into each of two 2.5 cm diameter × 2.5 cm deep depressions made into the soil. Once the 1.5 ml of inoculum is dispensed into each of the 2 depressions and absorbed into the soil, the holes are covered to prevent expulsion of the nematodes by subsequent watering. The plants are placed in the greenhouse, maintained at a temperature range of 25 °C-35 °C and given at least 12 h/day of ambient light supplemented with the cool white fluorescent lights, bringing the lighting to 16 h day/8 h night. The M. incognita life-stage development is described using a modified Christie's method (Christie 1946;Christie and Wash 1946;Tang et al. 1994). The nematodes are extracted by combined gravity screening and sucrose centrifugation at 50 days post infection (dpi). The nematodes are enumerated on grated Petri dishes with an Olympus BH2 B071 microscope (Japan Model C35AD-4) at 40 X magnification (Aljaafri et al. 2017).

Analysis of results
Root fresh weights are determined to allow the calculation of galls, egg masses, eggs, and J2s per gram of root tissue to standardize their presence in relation to the size (mass) of the root structure (see below) (Pant et al. 2016). The enumeration and statistical analyses of galls, egg masses, eggs, and J2s are done, analyzing them in two different ways. These two different analyses include enumerating the number of galls, egg masses, eggs, and J2s in relation to the whole root system (wr) which does not consider the effect the transgene has on root growth. A second analysis that does consider the effect that the transgene has on root growth is done by standardizing the number of galls and M. incognita per gram of root tissue (pg). In each analysis the results are considered statistically significant if p < 0.05, determined using Mann-Whitney-Wilcoxon Rank Sum Test (MWW) (Mann and Whitney 1947;Niraula et al. 2020a). The MWW Rank Sum Test is a nonparametric test of the null hypothesis not requiring the assumption of normal distributions (Mann and Whitney 1947).

Calculation of the reproductive factor (RF)
The RF is calculated as RF = eggs + juveniles extracted at 60 days post inoculation/2500. The denominator (2500) represents the starting inoculum of 2500 J2s. Direct comparison of the effect that the expression of MAPK3-1 has to MAPK3-2 is done using the Dunn's multiple comparisons test (Dunn 1964).

Analysis of MAPK3-1 and MAPK3-2 paralogs
The analysis begins by understanding the MAPK3-1 and MAPK3-2 paralogs better through an examination of their aa sequences. The analysis generates a 371 aa alignment having a 98.7% (366/371) identity and 98.9% (367/371) similarity with no gaps (Supplemental Fig. 1). At aa position 13 there is a P/A nonpolar to nonpolar R group difference, position 15 has a T/V nonpolar to nonpolar R group difference, position 36 has a A/T nonpolar to nonpolar R group difference; position 45 has a V/I nonpolar to nonpolar R group difference while position 142 has a S/C polar, but neutral to polar, but neutral R group difference. MAPK3-1 and MAPK3-2 have the TEY activation loop (A-loop) conserved domain (cd07858) at aa positions 197-199, consistent with plant MAPK3s.
Understanding the similarities and differences in MAPK3-1 and MAPK3-2 aa composition is important because the their proteins regulate the expression of proven defense genes occurring in common to them, and those regulated uniquely between them (McNeece et al. 2019; Klink et al. 2021a). RNA seq data for the MAPK3-1 and MAPK3-2 OE and RNAi roots and their respective pRAP15-ccdB (OE) Ontologies, specifically molecular function, are retrieved from Phytozome, using the PhytoMine tool (https:// phyto zome. jgi. doe. gov/ phyto mine/ begin. do) (Goodstein et al. 2012). Graphs are generated using Excel Table 1 Presented are gene counts for the MAPK3-1-OE and MAPK3-1-RNAi roots compared to their pRAP15-ccdB (overexpression) and pRAP17-ccdB (RNAi) control, as well as the MAPK3-2-OE and MAPK3-2-RNAi roots as compared to their pRAP15-ccdB and pRAP17-ccdB control, and genes in common between the two  Table 4, 5). A list limited to the top 10 most highly induced or suppressed genes occurring in common between the MAPK3-1-OE and MAPK3-2-OE roots in comparison to the pRAP15-ccdB control is presented (Table 3). A list limited to the top 10 most highly induced or suppressed genes occurring in common between the MAPK3-1-RNAi and MAPK3-2-RNAi roots as compared to the pRAP17-ccdB control is presented (Table 3). A complete gene list of the induced or suppressed genes expressed in the MAPK3-1-OE and MAPK3-2-OE roots in comparison to the pRAP15-ccdB control, as well as the MAPK3-1-RNAi and MAPK3-2-RNAi roots to the pRAP17-ccdB control is presented (Supplemental Tables 6 and  7). GO analyses presented here using RNA seq data obtained from MAPK3-1-OE or MAPK3-1-RNAi roots as compared to their pRAP15-ccdB or pRAP17-ccdB controls, respectively ( Fig. 1; Supplemental Tables 2, 3). The same comparisons are made for MAPK3-2-OE or MAPK3-2-RNAi roots as compared to the pRAP15-ccdB or pRAP17-ccdB controls, respectively ( Fig. 2; Supplemental Tables 4 and 5). GO analyses of induced or suppressed genes existing in common between the MAPK3-1-OE and MAPK3-2-OE lists in comparison to their pRAP15-ccdB control lists, and MAPK3-1-RNAi and MAPK3-2-RNAi lists in comparison to their pRAP17-ccdB control lists are provided ( Fig. 3; Supplemental Tables 6 and  7).
The high degree of sequence identity occurring between the G. max and G. hirsutum MAPK protein sequences lead to the hypothesis that the heterologous expression of the G. max MAPK3s in G. hirsutum will result in suppressing M. incognita parasitism. Furthermore, any significant differences in outcome occurring between the heterologous expression of MAPK3-1 and MAPK3-2 is limited to a relatively small number of nucleotides. This is because the expression of each gene is driven by the same FMV-Sgt promoter.
MAPK3-1 and MAPK3-2 can be expressed in G. hirsutum roots G. hirsutum is employed for a hairy root transgenesis procedure with the objective of expressing (E) the MAPK3-1-E or MAPK3-2-E cassettes in their roots using the pRAP15 expression plasmid (Supplemental Fig. 3) (Matsye et al. 2012). If needed, an RNA interference (RNAi) plasmid (pRAP17) was available (Klink et al. 2009). Steps in the hairy root procedure are presented (Supplemental Fig. 5) (Pant et al. 2015). Prior analyses show in rare cases that certain genes may not be able to undergo engineered expression, leading to the failure to obtain transgenic roots butthe results presented here show that G. hirsutum is expressing the MAPK3-1-E or MAPK3-2-E cassettes ( Fig. 4) (Austin et al. 2019). These genetically mosaic, composite plants have the entire shoot being non-transgenic while the entire root system is transgenic (Tepfer 1984;Haas et al. 1995;Collier et al.

MAPK3-1 and MAPK3-2 expression in G. hirsutum suppresses M. incognita gall production
M. incognita derives its nourishment from giant cells that are contained within an enlarged root structure called a gall, permitting an estimation of successful parasitism in many species of plants including G. hirsutum. Experiments presented here show that gall production normally induced by M. incognita is suppressed in G. hirsutum roots expressing the MAPK3-1-E or MAPK3-2-E cassettes as compared to the pRAP15-ccdB control in analyses of the whole root (wr) system (Fig. 5). To standardize the results, accounting for any developmental effects exerted on the G. hirsutum roots by the expression of the MAPK3-1 or MAPK3-2 transgenes, the number of M. incognita-induced galls occurring per gram (pg) of root tissue are calculated as compared to the pRAP15-ccdB control, also showing a reduction in gall number; p < 0.05, MWW Rank Sum Test. (Fig. 5). The effect that the expression of the MAPK3-1-E as compared to MAPK3-2-E cassettes has on gall production is not significantly different from each other in wr (p = 0.3492) and pg (p > 0.9999) analyses, Dunn's multiple comparisons test.

MAPK3-1 and MAPK3-2 expression in G. hirsutum suppresses M. incognita egg mass production
Analyses are performed here to assess sexual maturity by determining the number of egg masses that are made by female M. incognita. The analyses show that the production of egg masses is also suppressed in G. hirsutum roots engineered with the MAPK3-1-E or MAPK3-2-E cassettes in wr and pg analyses  as compared to the pRAP15-ccdB control; p < 0.05, MWW Rank Sum Test (Fig. 6). The effect that the expression of the MAPK3-1 as compared to MAPK3-2 transgenes has on egg mass production is not significantly different from each other in wr (p = 0.5228) and pg (p > 0.9999) analyses, Dunn's multiple comparisons test.

MAPK3-1 and MAPK3-2 expression in G. hirsutum does not negatively affect M. incognita egg production
In contrast to the results obtained for the number of galls and egg masses, the analyses reveal an increase in the number of M. incognita eggs in each of the roots genetically transformed with the MAPK3-1-E or MAPK3-2-E cassettes for the wr and pg analysis Ontologies, specifically molecular function, are retrieved from Phytozome, using the PhytoMine tool (https:// phyto zome. jgi. doe. gov/ phyto mine/ begin. do) (Goodstein et al. 2012). Graphs are generated using Excel  (Fig. 8). The effect that the expression of the MAPK3-1 as compared to MAPK3-2 transgene has on J2 production is not significantly different from each other in wr (p = 0.2104) and pg (p = 0.89) analyses, Dunn's multiple comparisons test. An added analysis is performed to determine whether reproduction of M. incognita is detrimentally affected by the genetic engineering of the MAPK3-1 or MAPK3-2 transgenes into G. hirsutum as compared to the controls, respectively. The analysis is accomplished by calculating the RF where RF = eggs + juveniles extracted at 60 days post inoculation/2500 (J2 inoculum) (please refer to the Materials section for details). The analysis identifies an RF of 0.65 for G. hirsutum roots engineered with the MAPK3-1 transgene and an RF of 0.81 for roots engineered with the MAPK3-2 transgene. In contrast, the pRAP15-ccdB control RF is 1.6412. An RF > 1.0 indicates reproduction is occurring. The RF is calculated using the total number of eggs and J2s extracted from the soil and therefore an RF per gram of root analysis is not performed. The results show the increase in egg number is balanced off by a sharp decrease in the number of J2s.

MAPK3-1 and MAPK3-2 expression in G. hirsutum affects its root growth
Analyses of the mass of G. hirsutum roots expressing the MAPK3-1-E or MAPK3-2-E cassettes as compared to the pRAP15-ccdB control reveal a significant decrease in root mass; p < 0.05, MWW Rank Sum Test (Fig. 9). This result explains the differences occurring for M. incognita-induced galls, egg masses, eggs, and J2s that are observed between the wr and pg MAPK3-1-E and MAPK3-2-E analyses in comparison to their pRAP15-ccdB controls, respectively. However, the effect that the expression of the MAPK3-1-E as compared to MAPK3-2-E cassette has on root development, in relation to their pRAP15-ccdB control, is not significantly different from each other (p = 0.1983), Dunn's multiple comparisons test.

MAPK3 homologs present in agriculturally important crops
The ability to heterologously express the MAPK3-1 and MAPK3-2 paralogs in G. hirsutum, leading to a defense response to M. incognita, indicates that the genes may function broadly in other plant species. Analyses of crops that are economically important worldwide and affected by climate change would aid from such transgenic studies like those done for G. max (Tilman et al. 2011;Liu et al. 2011;Neupane et al. 2013;Mohanta et al. 2015;Burkhead and Klink 2018;McNeece et al. 2019;Ray et al. 2013Ray et al. , 2019. MAPK homologs, including MAPK3, are identified

MAPK3 is an important defense node
The MAPK3-1 and MAPK3-2 are shown to be 98.7% identical to each other. When BLASTing the G. hirsutum genome with the MAPK3-1 and MAPK3-2 protein sequences, the same 6 G. hirsutum paralogs with 3 from its A genome and 3 from its D genome having 79-85% identify are identified. The high level of primary aa sequence conservation, along with the presence of the TEY A-loops lead to the hypothesis that their expression would likely produce a defense response to M. incognita parasitism in G. hirsutum. The RNA seq analyses identify defense genes that are induced or suppressed in expression. Recent experiments have examined a very narrow group of 309 G. max genes that are induced in their expression in each of 9 defense MAPK-OE root systems, including the overexpressed MAPK2, MAPK3-1, MAPK3-2, MAPK4-1, MAPK5-3, MAPK6-2, MAPK13-1, MAPK16-4, and MAPK20-2 as compared to the pRAP15-ccdB control (Niraula et al. 2020b). These genes have been further compared to respectively. Significance determined using Mann-Whitney-Wilcoxon Rank Sum Test (Mann and Whitney 1947). The number of experimental replicates, spanning the 3 biological replicates, include 30 MAPK3-1-E roots, 40 MAPK3-1-E roots, and 30 total pRAP15-ccdB-E roots. Please refer to Materials and Methods section, subsection: G. hirsutum genetic transformation, for details Fig. 9 Root growth in relation to MAPK3 (MK3) expression. Root growth is calculated as a percent with MK3-1 and MK3-2 average fresh weight divided by the pRAP15-ccdB control average fresh weight multiplied by 100. In each analysis the results are considered statistically significant if p < 0.05, determined using Mann-Whitney-Wilcoxon Rank Sum Test (Mann and Whitney 1947). A. Total change in root mass. B. Transformed data From A showing percent change in J2s as compared to the control. *, **, and *** denote statistical significance at the 0.05, 0.01, 0.001 probability level, respectively. The number of experimental replicates, spanning the 3 biological replicates, include 30 MAPK3-1-E roots, 40 MAPK3-1-E roots, and 30 total pRAP15-ccdB-E roots. please refer to Materials and Methods section, subsection: G. hirsutum genetic transformation, for details syncytium-expressed genes identified from two different H. glycines-resistant genotypes undergoing their defense responses (Niraula et al. 2020b). The cross comparison of these MAPK-OE and syncytium-expressed gene lists results in the identification of 8 putatively secreted proteins occurring in common between these studies having a defense function (Niraula et al. 2020b).
The GO analyses generate much larger lists of genes showing their understood biological role(s). These genes can be used in functional transgenic studies as done by Niraula et al. (2020b) to determine whether they exhibit a defense role. The MAPK3-1-OE and MAPK3-2-OE lists have 41.3% and 37.6% induced genes in common, respectfully. Analyses presented here show the MAPK3-1 and MAPK3-2 can induce the expression of genes like several polygalacturonase inhibiting protein 1 (PGIP1) paralogs (Glyma.05G123700, Glyma.08G078900, Glyma.05G123900), and the AtPEPR1 PRR receptor (Glyma.10G195700) that function in defense (Liu et al. 2013;Li and Smigocki 2018;Jing et al. 2020). In A. thaliana PEPR1 recognizes short peptides, leading to the activation of BRI-ASSOCIATED KINASE 1 (BAK1) and BOTRYTIS INDUCED KINASE1 (BIK1) to promote defense responses through MAPK signaling. In Pennisetum glaucum (pearl millet), its MAPK4 (PgMPK4) gene functions to induce the expression of the PGIP defense gene (Melvin et al. 2015). A B. vulgaris (sugar beet) PGIP expressed in Nicotiana benthamiana (tobacco) functions effectively in limiting the pathogenicity of Rhizoctonia solani, Fusarium solani, and Botrytis cinerea driven by their polygalacturonases (PGs) (Li and Smigocki 2018). The results presented here provide confidence that transgenic expression of the G. max MAPKs in G. hirsutum may function effectively in driving a defense response to M. incognita parasitism.

MAPK3-1 and MAPK3-2 can be expressed in G. hirsutum
The functional genetic engineering experiments succeeded in generating transgenic roots in G. hirsutum that are heterologously expressing the MAPK3-1 and MAPK3-2 genes. The ability to obtain transgenic roots that are expressing the target gene is not guaranteed. Austin et al. (2019) examined a family of syncytium-expressed myosin XI genes including Glyma.06G056500, Glyma.13G281900, Glyma.17G051900, Glyma.19G170700, and Glyma.20G001300, targeting them for overexpression in G. max. In A. thaliana, myosin XI functions in plant defense in processes involving vesicle transport and callose deposition (Yang et al. 2014). The overexpression of the myosin XI genes never led to the production of transgenic roots while control pRAP15-ccdB roots and roots targeted for overexpression of other genes could be obtained (Austin et al. 2019). In contrast, RNAi for the targeted myosin genes did generate roots with suppressed expression of the targeted gene and an increase in H. glycines parasitism while altering callose deposition (Austin et al. 2019The production of transgenic G. hirsutum expressing the MAPK3-1 and MAPK3-2 are produced successfully here, examining their potential role in defense to M. incognita.

MAPK3-1 and MAPK3-2 expression in G. hirsutum suppresses M. incognita gall production
The expression of MAPK3-1 leads to an 80.32% reduction in the production of galls in analyses of the whole root system, while there is a 68.11% reduction in the production of galls in analyses of the galls per gram of root system. The expression of MAPK3-2 leads to an 73.46% reduction of the production of galls in analyses of the whole root system, while there is a 66.88% reduction of the production of galls in analyses of the galls per gram of root system. MAPK3 functions downstream of NDR1 and harpin in ETI, making comparisons to our prior experiments expressing NDR1-1 in G. hirsutum relevant (Desikan et al. 1999;Knepper et al. 2011;Lang et al. 2021). Our prior results show the expression of NDR1-1 in G. hirsutum leads to a 70.7% reduction in gall production in whole root system analyses, while a 71.03% reduction in galls per gram of root system is observed . However, visibly obvious galls are not a prerequisite for M. incognita parasitism in some plants, so it is not an ideal marker for factors that negatively impact M. incognita parasitism.. incognita is to examine their effect on egg mass production. The expression of MAPK3-1 leads to an 82.37% reduction in the production of egg masses in analyses of the whole root system, while there is a 68.78% reduction in the production of egg masses in analyses per gram of root system. The expression of MAPK3-2 leads to a 76.79% reduction in the production of egg masses in analyses of the whole root system, while there is a 69.69% reduction in the production of egg masses in analyses per gram of root system. The expression of G. max NDR1-1 in G. hirsutum leads to a 53% reduction in egg mass production in whole root system analyses, while a 58.27% reduction in egg masses per gram of root system are observed . Therefore, the heterologous expression of MAPK3-1 or MAPK3-2 individually is more effective in generating a defense response than NDR1-1 is on its own. The results indicate that signals from more than one signaling pathway, perhaps PTI, converge on MAPK3 (Asai et al. 2002;Desikan et al. 2002;McNeece et al. 2019). In G. max, the overexpression of PTI components functioning upstream of MAPK3, including BAK1-1 and BIK1-6, along with the ETI component NDR1 all induce MAPK3 expression, leads to defense gene expression (Pant et al. 2014;McNeece et al. 2017McNeece et al. , 2019Klink et al. 2021a).

MAPK3-1 and MAPK3-2 expression does not suppress M. incognita egg production
The expression of MAPK3-1 leads to a 1.29-fold increase in egg production in analyses of the whole root system, while there is a 2.27-fold increase in egg production in analyses of the eggs per gram of root system. Similarly, the expression of MAPK3-2 leads to a 1.57-fold increase in the production of egg masses in analyses of the whole root system, while there is a twofold increase in the production of egg masses in analyses per gram of root system. The expression of G. max NDR1-1 in G. hirsutum leads to a 66.9% reduction in egg production in whole root system analyses, while a 73% reduction in eggs per gram of root system is observed . These observations lie in contrast to the results obtained for both MAPK3-1 and MAPK3-2 expression in G. hirsutum where an increase in eggs is observed.
The results indicate that there may be aspects of G. hirsutum gene signaling pathways that lie upstream of MAPK3-1 and MAPK3-2, also negatively impacting M. incognita egg production, that are not engaged by the expression of MAPK3 which functions downstream of NDR1 (Qin et al. 2018). Alternatively, other functions, including downstream processes are involved (Li et al. 2016).
MAPK3-1 and MAPK3-2 expression suppresses M. incognita J2 production Experiments show that the expression of G. max MAPK3-1 leads to an 88.21% reduction in the production of J2s in analyses of the whole root system, while there is a 76.98% reduction of the production of J2s in analyses per gram of root system. The expression of G. max MAPK3-2 leads to an 84.07% reduction of the production of J2s in analyses of the whole root system, while there is a 66.88% reduction of the production of J2s in analyses per gram of root system. In comparison to our prior results, the expression of G. max NDR1-1 in G. hirsutum leads to a 60.67% reduction in J2 production in whole root system analyses, while a 66.57% reduction in eggs per gram of root system is observed . These observations made for the expression of G. max NDR1-1 in G. hirsutum exhibits similarity with the results obtained for both MAPK3-1 and MAPK3-2 expression in G. hirsutum.
MAPK3-1 and MAPK3-2 expression in G. hirsutum decreases the M. incognita reproductive factor MAPK3 functions as a signaling node, receiving input from PTI and ETI defense branches (Desikan et al. 1999;Yi et al. 2015;McNeece et al. 2019;Yuan et al. 2021;Lang et al. 2021). Experiments presented here demonstrate a significant, negative effect that the expression of the 2 MAPK3 genes have on M. incognita gall, egg mass, and J2 production in G. hirsutum (Fig. 10). Prior experiments examining whether the expression of genes exert their effects on M. incognita development in G. hirsutum have examined the PTI-regulated NPR1 (G. max NPR1-2), ETI regulated NDR1 (G. max NDR1-1), and two secreted genes including a g and XTH (G. max g-4 and XTH43 (Fig. 10). The G. max NDR1-2 overexpression in G. max increases the relative transcript abundance of soybean NPR1-2, g-4, and XTH43 (McNeece et al. 2017).
The results for the M. incognita-induced gall studies show that the MAPK3-1-E (68.11% reduction), and MAPK3-2-E (66.88% reduction) root systems are about as effective as the G. max NDR1-1-E (71.03% reduction), and NPR1-2-E (66.01% reduction) root systems in decreasing gall number (Fig. 10). However, the expression of the G. max secreted g-4 (56.97% reduction), and XTH43 (17.7% reduction) are less effective. The similarity in outcome of MAPK3-1 and MAPK3-2 to those obtained for NDR1-1 and NPR1-2 are consistent with the proteins functioning in the same genetic pathway. Harpin signals through NDR1 and MAPK, leading to a defense response (Gopalan et al. 1996;Desikan et al. 1999Desikan et al. , 2001Lee et al. 2001;McNeece et al. 2019;Lang et al. 2021). In G. max, NDR1-1 overexpression leads to the induced expression of MAPK3-2 (which leads to the induced expression of RO-40, TGA2-1, SHMT-5, and NPR1-1), MAPK20-2 (which leads to the induced expression of TGA2-1, EDS1-1, , and PR1-6) (Falk et al. 1999;McNeece et al. 2019). In G. max, the NPR1-2 overexpression cassette induces its own expression (NPR1-2) while also increasing the relative transcript abundances of XTH43, BIK1-6, the salicylic acid regulated secreted protein gene PR1-6, the ethylene and jasmonic acid responsive secreted protein gene basic chitinase PR3 (Glyma.02G042500), the rhg1 locus component amino acid transporter (AAT ) (Glyma.18G022400), and SHMT-5 (Antoniw and Pierpoint 1978;Legrand et al. 1987;Liu et al. 2012;Pant et al. 2014).. In these analyses MAPK3-1-E and MAPK3-2-E engineered in G. hirsutum suppresses M. incognita egg mass production by 68.78% and 66.88%, respectively (Fig. 10). NDR1-1-E engineered in G. hirsutum suppresses egg mass production by 58.27%, while NPR1-2-E engineered in G. hirsutum suppresses M. incognita development by 68.18%, levels similar as MAPK3-1-E and MAPK3-2-E. The secreted g-4-E engineered in G. hirsutum suppresses M. incognita by 77.4%, while XTH43-E engineered in G. hirsutum suppresses M. incognita by 70%. Generally, each of these genes are functioning at levels that are similar, with highly effective suppression of egg mass production occurring. The disparity between gall production and egg mass formation in g-4-E and XTH43-E G. hirsutum is striking. The results indicate that those genes are not as important for the impairment of gall formation, showing that defense processes targeting gall and syncytium formation do exhibit specificity (Pant et al. 2014;Niraula et al. 2020b). M. incognita egg masses are structures that contain the eggs and a gelatinous matrix. A surprise that came out of the analysis of egg quantity is that while MAPK3-1 and MAPK3-2 expression leads to a significant decrease (> 66%) in egg mass production, the number of eggs increases significantly by 2.27-fold and twofold, respectively. In contrast, NDR1-1, and NPR1-2 expression in G. hirsutum roots decreases egg production by 73% and Fig. 10 Comparison of the percent effect that G. max MAPK3-1 or MAPK3-2 expression has on M. incognita gall, egg mass, eggs and J2 production to prior analyses of NPR1-2, NDR1-1, g-4, and XTH43. *, ** NPR1-2, g-4 (Pant et al. 2016), *** NDR1-1 (McNeece et al. 2017), **** XTH43 (Niraula et al. 2020a) 77.55%, respectively. The experimentally-expressed g-4 and XTH43 in G. hirsutum is also highly effective in suppressing M. incognita egg production, leading to a 77.55% and 79.55% decrease, respectively. It is unclear why there would be such a significant increase in the amounts of eggs in the MAPK3-1 and MAPK3-2-expressing G. hirsutum roots. In a process known as hormesis, altered hormone concentrations in the dipteran insect Bactrocera dorsalis occurring by exposure to a low, sublethal concentration of a synthetic anthranilic diamide ryanodine receptor agonist insecticide cyantraniliprole leads to an increase in egg production (Zhang et al. 2015). Furthermore, sublethal doses of plant secondary metabolites can also increase insect fecundity so the effects are not limited to synthetic, non-plant compounds (Papanastasiou et al. 2017). Similar results have been observed in nematodes (Martel et al. 2020). The results presented here indicate that a very specific process is occurring in G. hirsutum due to the altered transcriptional program caused by the MAPK3-1 and MAPK3-2 expression. Their expression in G. hirsutum is leading to the production or absence of a bioactive molecule(s) that produce contrasting impacts, both negative (decreased gall, egg mass, and J2s) and positive (increased egg production) on M. incognita. While an increase in M. incognita egg production is observed, it is possible that the eggs are inviable and that a mechanism by which M. incognita produces viable J2s is impaired in G. hirsutum roots expressing MAPK3-1 and MAPK3-2. Having the expression of each MAPK3 paralog leading to the same outcome across multiple replicates argues against the result being nonspecific. To examine this observation further, an analysis of J2s is performed.
The analysis of M. incognita J2 quantity results in the demonstration that the expression of MAPK3-1 and MAPK3-2 in G. hirsutum leads to a 76.98% and 66.57% decrease, respectively. The expression of NDR1-1 and NPR1-2 in G. hirsutum leads to a 66.57% and 88.58% decrease in J2s, respectively, while the experimentally-induced g-4 and XTH43 gene expression in G. hirsutum reduces M. incognita J2s by 91.65% and 96.8%, respectfully. What the results indicate for MAPK3-1 and MAPK3-2-expressing G. hirsutum roots is that a significant amount of M. incognita eggs could be non-viable. In contrast, the NDR1-1 and NPR1-2-expressing G. hirsutum roots have J2 values similar with what is observed for galls (71.03% and 66.01% reduction, respectively), egg masses (58.27% and 68.18% reduction, respectively), and eggs (73% and 77.55% reduction, respectively). These results indicate that the effects exerted on M. incognita by G. max NDR1-1 and NPR1-2 expression in G. hirsutum occur early during infection and remain in place throughout the plant-nematode interaction during gall, egg mass, egg, and J2 production. The gene expression results for the G. max g-4 and XTH43 differ in that there appears to be a cumulative negative effect that their expression in G. hirsutum has on M. incognita (Fig. 10). A cumulative negative effect on pathogen fitness is observed for transgenic plants expressing-glucosidases (Zagrobelny et al. 2007(Zagrobelny et al. , 2008. The trends seen for the effects generated by heterologous gene expression for the examined genes in relation to M. incognita galls, egg masses, eggs, and J2s demonstrate that it is possible to target specific aspects of the pathogen life cycle (Scheideler et al. 2002). However, the expression of multiple genes may aid in producing an additive effect that could eliminate infection or parasitism altogether (Zhang and Shapiro 2002;Zhou et al. 2020).
The results presented here show both G. max g-4 and XTH43 expression function highly effectively in mitigating M. incognita as demonstrated by their 91.65 and 96.8% reduction in J2 production, respectively (Pant et al. 2016;Niraula et al. 2020b). These results are consistent with the effect that MAPKinduced, syncytium-expressed secreted proteins have on H. glycines development in G. max (Niraula et al. 2020b). It is clear from these observations that it is possible to obtain a very high suppression of M. incognita development through the expression of genes from heterologous sources. Using promoters that effectively drive that expression in the presence of the nematode which has a significant capacity to control root cell gene expression is an important consideration when studying how genes function during pathogenesis and defense (Klink et al. 2009(Klink et al. , 2021aAli and Kim 2019). The analysis presented here describes the effect that the MAPK3-1 and MAPK3-2 expression has on M. incognita parasitism in G. hirsutum and discussing those data in relation to the previously studied NDR1-1, NPR1-2, g-4, and XTH43 (Pant et al. 2016;McNeece et al. 2017;Niraula et al. 2020b). A comparison of the results obtained for those genes demonstrates that, unlike the G. max MAPK3-1, MAPK3-2, NDR1-1, and NPR-1-2 signaling genes, the heterologous expression of g-4 and XTH43 secreted protein genes clearly have a cumulative negative effect on M. incognita at later stages of its life cycle, in particular J2s (Fig. 10). The results show that there are genes functioning effectively at different stages of the defense response that require further exploration as to what their role(s) are. For example, cell wall biochemical analyses show the G. max XTH43 overexpression shortens XyG chain length, increases the number of those shorter XyG chains and increases the amount of XyG, consistent with earlier cytological and ultrastructural studies of syncytia undergoing a defense response (Ross 1958;Endo 1965Endo , 1991Niraula et al. 2021). XTH43-RNAi has the opposite effect (Niraula et al. 2021).
The increase in the number of shorter chains may interfere with a secreted pathogen effector's ability to enzymatically degrade cell walls by producing a diffusion barrier (Niraula et al. 2020b). Alternatively, the nematode does not produce enough walldegrading enzyme to combat the extra wall material it encounters.
MAPK3-1 and MAPK3-2 expression in G. hirsutum affects its root growth Differences are observed in the relative amount of M. incognita-induced galls, egg masses, eggs, and J2s in analyses of data obtained from MAPK3-1-E and MAPK3-2-E whole root systems as compared to the numbers per gram of root system, in relation to the pRAP15-ccdB control roots. Therefore, the expression of the MAPK3-1 or MAPK3-2 transgenes in G .   Fig. 11 Model. G. max ETI and PTI genes expressed in G. hirsutum lead to a defense response. These functioning genes include the ETI gene NDR1 which acts with RIN4, RPM1, and RPS2, whose expression leads to MAPK expression and downstream gene expression including the expression of the secreted genes g-4, and XTH43. PTI genes including PRRs, the co-receptor BAK1, the associated cytoplasmic kinase BIK1 lead to induced EDS1 and NPR1 expression. PTI and ETI cross communicate. EDS1 signals through NPR1 to activate the transcription of downstream genes functioning in defense. G. max genes denoted with hashed lines (i.e., EDS1, BIK1, RIN4, RPM1, RPS2) have not been heterologously expressed in G. hirsutum. Genes with solid lines (i.e., MAPK3-1, XTH43) have been heterologously expressed in G. hirsutum (Yi et al. 2015;Pant et al. 2016;McNeece et al. 2017;Chen et al. 2017;Liu et al. 2020;Yuan et al. 2021;Niraula et al. 2020a, b) hirsutum affects root mass as compared to the respective controls, in these cases negatively. The expression of the G. max XTH43, NPR1-2 or g-4 in G. hirsutum leads to no statistically significant effect on root mass (Pant et al. 2016;Niraula et al. 2020a). XTH43 and g-4, as secreted proteins would be expected to function downstream of MAPK signaling. NPR1-2 functions as a co-transcriptional regulator with TGA2-1 and would be expected to function downstream of MAPK3. Therefore, targeting downstream genes that encode the secreted XTH43 and g-4 may be an effective way to generate resistance while not experiencing the drag of reduced root mass.
Targeting of MAPK signaling in understudied, but agriculturally relevant crops, for defense Significant agricultural plant species are presented in recent studies, (Tilman et al. 2011;Ray et al. 2013Ray et al. , 2019. Relating to climate change (). While MAPKs are studied under certain circumstances, in some crops, they are not annotated. The analysis presented here in those cases identify their MAPKs to aid in the analysis of these genes in relation to plant defense and other basic biological roles including climate change (Burkhead and Klink 2018;Li and Smigocki 2018). ().

Model
The experiments presented here show it is possible to genetically engineer in an important node (MAPK) functioning in plant defense signaling acting downstream of the PTI and ETI receptors. The effort has resulted in the generation of a highly resistant reaction,impairing M. incognita development (Fig. 11) (Yi et al. 2015;Chen et al. 2017;Niu et al. 2016;Nie et al. 2017;McNeece et al. 2017;Aljaafri et al. 2017).