Exocyst components promote an incompatible interaction between glycine max (soybean) and heterodera glycines (the soybean cyst nematode)

ABSTRACT Vesicle and target membrane fusion involves tethering, docking and fusion. The GTPase _SECRETORY4_ (_SEC4_) positions the exocyst complex during vesicle membrane tethering,

facilitating docking and fusion. _Glycine max_ (soybean) Sec4 functions in the root during its defense against the parasitic nematode _Heterodera glycines_ as it attempts to develop a

multinucleate nurse cell (syncytium) serving to nourish the nematode over its 30-day life cycle. Results indicate that other tethering proteins are also important for defense. The _G. max_

exocyst is encoded by 61 genes: 5 EXOC1 (Sec3), 2 EXOC2 (Sec5), 5 EXOC3 (Sec6), 2 EXOC4 (Sec8), 2 EXOC5 (Sec10) 6 EXOC6 (Sec15), 31 EXOC7 (Exo70) and 8 EXOC8 (Exo84) genes. At least one

member of each gene family is expressed within the syncytium during the defense response. Syncytium-expressed exocyst genes function in defense while some are under transcriptional

regulation by mitogen-activated protein kinases (MAPKs). The exocyst component EXOC7-H4-1 is not expressed within the syncytium but functions in defense and is under MAPK regulation. The

tethering stage of vesicle transport has been demonstrated to play an important role in defense in the _G. max_-_H. glycines_ pathosystem, with some of the spatially and temporally regulated

exocyst components under transcriptional control by MAPKs. SIMILAR CONTENT BEING VIEWED BY OTHERS THE SOYBEAN PLASMA MEMBRANE GMDR1 PROTEIN CONFERRING BROAD-SPECTRUM DISEASE AND PEST

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Article Open access 30 March 2022 INTRODUCTION During their defense against pathogen infection, plants employ cellular processes to detect and amplify signals derived from the activities of

those pathogens. If successful, these plant processes lead to resistance1. Among the better known defense processes that lead to resistance are those mediated by resistance (R) proteins that

function as pattern recognition receptors (PRRs)1. PRRs themselves function within the context of effector-triggered immunity (ETI) and pathogen-associated molecular pattern-triggered

immunity (PTI) processes1,2,3,4,5. Additionally, among these defense proteins is a cellular apparatus that functions in vesicle transport6,7. Upon its secretion, this vesicle transport

apparatus delivers cargo that inhibits pathogen infection6,7. The apparatus also delivers and internalizes PRRs, facilitating defense6,7. Through a genetic approach, Novick et al_._8

analyzed the vesicle transport apparatus using the ascomycete fungus _Saccharomyces cerevisiae_. In generating secretory (_sec_) mutants, Novick et al_._8 identified genes whose protein

products are responsible for vesicle transport. Unrelated experiments using the plant genetic model _Arabidopsis thaliana_ have aided in the identification of PENETRATION1 (PEN1)6,9,10,11.

PEN1 is a syntaxin and the first protein among plant vesicle transport protein homologs shown to function in defense6,11. Consequently, this work revealed that vesicle transport proteins are

universal among eukaryotes and important to the defense process9,10. Syntaxin functions as part of a membrane receptor complex known as soluble N-ethylmaleimide-sensitive fusion protein

attachment protein receptor (SNARE). SNARE itself is part of an even larger complex known as the 20 S particle. The 20 S particle was identified through work on _Rattus norvegicus_ (rat)

liver tissue using purified recombinant human N-ethylmaleimide-sensitive fusion protein (NSF) and α-soluble N-ethylmaleimide-sensitive fusion protein attachment protein (α-SNAP)12,13,14.

Other PEN proteins in plants have been identified, including PEN2 (a β-glucosidase) and PEN3 (an ATP-binding cassette [ABC] transporter), which function together as a more broadly associated

unit called the regulon6,15,16,17. Therefore, the regulon consists of numerous proteins, including 20 S particle components that act together during the defense response. Experiments have

focused on examining shoot pathogens, but studies examining root pathogens are largely lacking6,15,16,18,19. Among the many important root pathogens are plant parasitic nematodes, a

devastating group among a much larger cohort of organisms that have significant global effects20. Recent experiments have examined the function of regulon proteins in plant roots in relation

to root pathogens. These experiments employed the interaction between _Glycine max_ (soybean) and the plant parasitic nematode _Heterodera glycines_ (the soybean cyst nematode [SCN]) as a

model. _H. glycines_ is an obligate parasite, with _G_. _max_ its primary host, that causes billions of dollars in economic losses every year21,22. Furthermore, _H. glycines_ causes more

economic losses than other _G. max_ pathogens combined22. _G. max_ may show obvious signs of _H. glycines_ parasitism, such as chlorosis and stunting. However, complicating _H. glycines_

detection, _G. max_ may also show no adverse signs of parasitism except for an approximately 15% decrease in yield23. _H. glycines_ also consists of a number of genetically distinctive

variants, known as HG types, that relate in various ways to a race scheme24,25,26. Consequently, _G. max_ cultivars that normally exhibit natural resistance to _H. glycines_ can succumb to

infection through processes dependent on the HG type with which it is infected. _H. glycines_ has a life cycle of 30 or more days depending on ambient temperature27. Importantly, as part of

its life cycle, _H. glycines_ generates a hardened cyst formed from the carcass of the female containing 250–500 eggs. The eggs within these cysts can remain dormant in the soil for up to 9 

years, complicating management practices. During successful parasitism of _G. max_, _H. glycines_ eggs hatch as second-stage juveniles (J2s). The J2s migrate toward and then burrow into the

root, subsequently slicing through epidermal, cortex and endodermal root cells with a rigid, tubular mouth apparatus known as a stylet. Upon reaching the root stele, the J2s use the stylet

to deliver effectors into a _G. max_ pericycle or neighboring cell. Over a period of days, the cell walls of the _H. glycines_-parasitized root cells dissolve through enzymatically driven

processes mediated by the nematode. The outcome is the production of a multinucleate syncytium, the product of the incorporation of 200–250 neighboring root cells into a common

cytoplasm28,29. The syncytium is also the site of the localized defense response30, which involves components of ETI and PTI31,32,33. However, for the purposes here, the term defense is used

to refer to this localized defense response15. A _G. max_ ortholog of the 20 S particle vesicle transport apparatus component α-SNAP-5 (Glyma.18G022500) maps to the major _H. glycines_

resistance locus (_rhg1_). The defense function of α-SNAP-5 has been shown through functional transgenic experiments31. Here, functional, transgenic experimental approaches included

experimental increases in target gene expression through overexpression of the target gene in an _H. glycines_-susceptible _G. max_ cultivar (_G. max_[Williams 82/PI 518671]), suppressing

parasitism, and experimental decreases in target gene expression through RNA interference (RNAi) in an _H. glycines_-resistant _G. max_ cultivar (_G. max_[Peking/PI 548402]), facilitating

parasitism. The combination of experimentally suppressing _H. glycines_ parasitism in _G. max_[Williams 82/PI 518671] and experimentally facilitating _H. glycines_ parasitism in _G.

max_[Peking/PI 548402] has demonstrated the roles of targeted genes, such as α-SNAP-5, in defense31,32,33,34. Complimentary studies have shown that an _H. glycines_ effector directly binds

α-SNAP-535. Presumably, this nematode effector impairs or hijacks α-SNAP-5, interfering with its normal role during the _G. max_ defense response. In this manner, the _H. glycines_ effector

generates effector-triggered susceptibility (ETS)1. These observations are similar to those in original studies showing that microbial effectors impaired SNARE protein function in animal

systems using _R. norvegicus_ and _Aplysia californica_ (the sea slug) as models36. Those studies examining microbial pathogenesis showed that the microbial neurotoxin effectors botulinum

from _Clostridium botulinum_ and tetanus from _C. tetani_ target SNARE components36. The interaction between botulinum and tetanus effectors inhibits secretion, resulting in paralysis36.

These studies suggest that while _G. max_ has a functional membrane fusion apparatus that it employs to impair _H. glycines_ pathogenesis, nematode effectors target and perturb its function,

facilitating parasitism35. These _H. glycines_ effectors bind the _G. max_ vesicle transport protein, presumably altering its function for the benefit of the parasite in ETS35. The vesicle

and target membrane fusion process consists of three steps: vesicle tethering to a target membrane, docking, and subsequent fusion of the vesicle and target membrane. The successful

completion of this three-step membrane fusion process leads to the delivery of PRRs and release of the vesicular cargo. While components of the 20 S vesicle docking particle have been shown

to play roles in defense against a number of pathogens, including _H. glycines_, in _G. max_, much less regarding the upstream process of tethering is known. In _S. cerevisiae_, a

vesicle-bound protein known as Sec4p initiates tethering, and in _G. max,_ a Sec4 homolog functions in defense against _H. glycines_37. _S. cerevisiae_ Sec4p is a Rab GTPase that regulates

the assembly of a structure called the exocyst38,39. This function of Sec4p in mediating tethering occurs through its interaction with the exocyst component Sec15p38,39. These results

indicate that the _G. max_ exocyst also performs a defense function against _H. glycines_ parasitism. The exocyst is an octamer composed of Sec3p (EXOC1), Sec5p (EXOC2), Sec6p (EXOC3), Sec8p

(EXOC4), Sec10p (EXOC5), Sec15p (EXOC6), Exo70p (EXOC7) and Exo84p (EXOC8)38,40,41,42,43,44. The exocyst complex acts as a signal receiver for various signaling pathways44,45. Through this

role, the exocyst helps tether vesicles at the receptor membrane and mediate fusion by inducing SNARE assembly44,45. Thus, tethering occurs upstream of the roles of the SNARE-containing 20 S

particle in docking and fusion. The exocyst functions to promote a number of cellular processes. In general, these processes include exocytosis, cell polarity, growth, division, cell

migration, ciliogenesis, autophagy, pollen compatibility and plant defense8,18,44,45,46,47. Relevant to plant defense is exocytosis, an evolutionarily conserved biological process that

ultimately facilitates the fusion of secretory vesicles with a targeted membrane. Consequently, exocysts allow cells to deliver PRRs and cargo that may function in defense8,18,44,45,46,47.

Each exocyst component plays an important role in secretion. Mutants of _S. cerevisiae_ Sec3 (_sec3_), the primary exocyst subunit that connects vesicles with the target membrane, exhibit

secretory vesicle accumulation in the cytoplasm48,49 because vesicles are unable to tether with the target membrane48,49. _N. benthamiana_ Sec5 (EXOC2) is important for secretion of the

pathogenesis-related 1 (PR-1) protein and callose deposition in the process of defense against _Phytophthora infestans_18. _N. benthamiana_ Sec5 is targeted by the _P. infestans_ effector

AVR1 RXLR, which impairs PR-1 secretion and callose deposition18. Notably, _G. max_ PR-1 (Glyma.15G062400) functions in defense against _H. glycines_50. _G. max_ PR-1 is also under

regulation by mitogen-activated protein kinases (MAPKs)50. The work of Austin et al_._51 led to the identification of a number of _G. max_ callose synthases (CSs) expressed within the

syncytium during the defense process with functions in defense. The results demonstrated that a secreted _G. max_ protein (PR-1) and an enzyme (CS) that generates a secreted defense molecule

(callose) function during the defense response against _H. glycines_51. Therefore, _G. max_ PR-1 and CS act in a manner that is very similar to their function in _N. benthamiana_ during

PTI11. In another recent work, some pathogen effectors were shown to impair the function of the exocyst structure through ubiquitination of an exocyst protein component (Exo70B1) in a manner

resembling ETS52. Due to the importance of each exocyst component, experiments have shown that the removal of just one protein impairs the ability of the other components to function

properly52,53,54, resulting in the impairment of biological processes52,53,54. Exocyst proteins are coiled-coil proteins that share some structural homology with helical bundles41,55.

Helical bundles facilitate exocyst component interactions, which are essential for complex formation41,55. The structure of the exocyst complex is rod-shaped, with N- and C-termini located

at opposite poles of the structure. This structure aids in the tethering of vesicles to the plasma membrane and delivery of vesicle cargo to the apoplast38,40,44,46,56,57,58. The exocyst

functions by connecting vesicles through the EXOC5 and EXOC6 proteins to the plasma membrane through EXOC1 and EXOC738,44,49,59,60. On the target (plasma) membrane is phosphatidylinositol

4,5-biphosphate (PI(4,5)P2), to which EXOC1 and EXOC7 bind61,62,63. In _S. cerevisiae_, the movement of vesicles is regulated by vesicle membrane-bound Sec4p, which directs the vesicle to

the plasma membrane at a targeted site64,65,66,67. Through its interaction with EXOC6, Sec4p functions by regulating assembly of the exocyst38,39. These results support observations showing

that _G. max_ Sec4 functions in facilitating the defense response to _H. glycines_37. The experiments presented here have identified the components of the _G. max_ exocyst. At least one

exocyst component of each gene family is expressed within the syncytium during the defense response of _G. max_ against _H. glycines_ parasitism. In some cases, these exocyst genes are under

regulation by MAPKs. Experimental overexpression of exocyst genes in the _H. glycines_-susceptible cultivar _G. max_[Williams 82/PI 518671] suppresses parasitism. In contrast, experimental

decreases in the expression of exocyst components through RNAi in the _H. glycines_-resistant cultivar _G. max_[Peking/Pi 548402] facilitate parasitism. The combination of suppressing _H.

glycines_ parasitism in a normally susceptible _G. max_ cultivar and facilitating _H. glycines_ parasitism in a normally resistant _G. max_ cultivar successfully demonstrated the functions

of the target genes in defense. These results demonstrate that the _G. max_ exocyst plays an important role in defense against _H. glycines_ parasitism. Furthermore, these results show the

importance of the plant secretion process to defense in general. RESULTS EXOCYST GENES WERE EXPRESSED WITHIN _H. GLYCINES_-PARASITIZED ROOT CELLS DURING DEFENSE The observation that _G. max_

Sec4 functions in defense against _H. glycines_ parasitism implies a similar role for the exocyst37. The most recently released _G. max_ genome annotation (Wm82.a2.v1) was examined through

BLAST searches using _A. thaliana_ exocyst component protein sequences as a query. The analysis resulted in the identification of 5 EXOC1 genes, 2 EXOC2 genes, 5 EXOC3 genes, 2 EXOC4 genes,

2 EXOC5 genes, 6 EXOC6 genes, 31 EXOC7 genes and 8 EXOC8 genes (Supplementary Table S1). These gene accessions served as the basis for subsequent analyses. Here, the _H. glycines_ life cycle

guided the design of gene expression experiments (Fig. 1)31 employing LM to isolate RNA from targeted cells. The targeted cells are involved in successful parasitism by _H. glycines_ during

a susceptible reaction and the defense response by _G. max_ during a resistant reaction. The collected cells included pericycle cells and surrounding cells and were collected at 0 dpi.

Furthermore, syncytia were collected at an early stage of parasitism (3 dpi). Syncytia formed in susceptible or resistant reactions at 3 dpi showed a similar cytological appearance. Their

features included hypertrophy, the enlargement of nuclei, the development of dense cytoplasm and an increase in the endoplasmic reticulum (ER) and ribosome content. As a consequence of these

similarities, 6 dpi was selected as a time point. The 6-dpi time point assisted in differentiating between a susceptible and resistant reaction. By 6 dpi, the syncytia formed during a

susceptible reaction were characterized by the hypertrophy of nuclei and nucleoli, proliferation of cytoplasmic organelles, a reduction in vacuoles, the dissolution of vacuoles and cell

expansion due to the incorporation of adjacent cells. In contrast, the cytoplasmic characteristics of the resistant reaction were genotype-specific. For example, by 6 dpi, _G. max_[Peking/PI

548402] showed cell wall apposition, structures consisting of cytoplasmic components that aggregated through actin polarization and the vesicle-mediated delivery of cargo. Furthermore, the

_G. max_[Peking/PI 548402] defense response at 6 dpi included the production of a necrotic layer of cells surrounding the syncytium and the accumulation of ER, which led _H. glycines_

development to be blocked at the parasitic J2 stage. In contrast, the _G. max_[PI 88788] defense response was not characterized by cell wall apposition or a necrotic layer of cells

surrounding the syncytium during the resistance reaction, but the ER had accumulated, leading to the blockade of _H. glycines_ development at the J3–J4 stage (Fig. 1)31. The cDNA probe made

from mRNA31 was tagged with a proprietary Affymetrix label and used for gene expression studies, leading to the identification of a pool of 1,787 candidate defense genes (Fig. 1)31. This

analysis was focused on examining the relationship between _G. max_ and its defense response against _H. glycines_ parasitism in relation to the exocyst (Fig. 1). From these data, exocyst

genes expressed within the syncytium were identified (Fig. 2). Under our analytical parameters, the exocyst genes exhibited four profiles of expression in relation to their defense response

to _H. glycines_. However, other gene expression profiles not observed here are possible. First, 11 exocyst genes were not expressed at any time point: EXOC1-3, EXOC6-3, EXOC8-3, EXOC8-5,

EXOC8-8, EXOC7-A1-1, EXOC7-B1-1, EXOC7-B1-3, EXOC7-E2-1, EXOC7-F1-2 and EXOC7-H4-1 (Fig. 2). Second, 8 exocyst genes lacked measurable gene expression at 0 dpi (control) but were expressed

at the 6-dpi time point: EXOC1-1, EXOC3-5, EXOC4-1, EXOC6-1, EXOC6-6, EXOC7-D1-2, EXOC7-E1-1 and EXOC7-G1-4 (Fig. 2). The third group was composed of 4 exocyst genes expressed at only 3 and

6 dpi: EXOC5-2, EXOC8-4, EXOC7-B1-2 and EXOC7-H7-1 (Fig. 2). The fourth group of 4 exocyst genes was expressed at all three time points (0, 3 and 6 dpi): EXOC2-1, EXOC7-A1-3, EXOC7-D1-1 and

EXOC7-F1-1 (Fig. 2). Consequently, the results revealed that 16 different exocyst genes were expressed in samples from at least one of the time points chosen for the analysis. Furthermore,

the analyses identified a component of each exocyst gene family expressed within cells parasitized by _H. glycines_ as the root cell underwent a defense response. Last, among these 16

exocyst genes, four were expressed at the 0-dpi time point, 8 were expressed by the 3-dpi time point, and 16 were expressed by the 6-dpi time point. These results demonstrate an increase in

the number of exocyst genes expressed during the course of the defense process. Notably, expression of a number of exocyst genes in root cells could not be evaluated (n/a) due to the nature

of the original root cell gene expression analyses (Supplementary Table S2)31. While not studied in the functional analyses presented in “Functional analysis of syncytium-expressing exocyst

genes demonstrates a defense role” section, the expression of those exocyst genes was examined in transcriptomic analyses of defense MAPKs, the results of which are presented in “The

expression of certain exocyst genes was induced by specific defense MAPKs” section50. An additional examination determined whether the exocyst genes are regulated by signaling processes in

the _G. max_ defense against _H. glycines_ and may also be of interest here50. THE EXPRESSION OF CERTAIN EXOCYST GENES WAS INDUCED BY SPECIFIC DEFENSE MAPKS Recent experiments identified a

subset of 9 (of the 32) _G. max_ MAPKs whose experimentally induced expression in the normally _H. glycines_-susceptible cultivar _G. max_[Williams 82/PI 518671], resulted in an engineered

defense response to _H. glycines_50. In contrast, experimental suppression of the expression of the same 9 defense MAPKs by RNAi in the normally _H. glycines_-resistant _G. max_[Peking/PI

548402] cultivar impaired the defense response50. RNA from these defense MAPK-OE and RNAi transgenic lines was subjected to RNA-seq analyses68, which led to the identification of thousands

of transcripts whose relative abundances either increased or decreased68. Consequently, the _G. max_ exocyst gene family as a whole was examined here via transcriptomic analyses of those

MAPK-OE and MAPK-RNAi lines. Analyses of those RNA-seq data was conducted to determine if the syncytium-expressed exocyst genes was also expressed within individual MAPK-OE or MAPK-RNAi

lines50. Further, the analysis also determined whether the exocyst genes were expressed across many lines overexpressing the defense MAPKs50. The results demonstrated that the differential

expression of exocyst genes was primarily found in specific transgenic MAPK lines (Supplementary Table S2). For many exocyst genes, differential expression was not observed at all

(Supplementary Table S2). However, even if an exocyst gene was not differentially expressed (NDE), this does not mean that the gene is not expressed at all (lacking identified sequences in

the RNA-seq studies). Among the exocyst genes inducing MAPK overexpression, EXOC7-H4-1 and EXOC7-H7-1 exhibited higher relative transcript levels in all 9 defense MAPK-OE lines. However,

when their expression within the syncytium was examined, EXOC7-H4-1 lacked expression in the 0-dpi control samples as well as the 3-dpi and 6-dpi samples from syncytia during the defense

response (Fig. 2). While EXOC7-E2-1 was also not expressed in the 0-dpi control samples or 3-dpi or 6-dpi syncytium samples, was expressed at almost the same level as EXOC7-H4-1in the

defense MAPK-OE lines. However, EXOC7-E2-1 gene expression was observed in just 8 of the 9 defense MAPK-OE lines. EXOC7-E2-1 was not further examined via qRT-PCR or functional studies since

it was not expressed in the syncytium or in all 9 of the defense MAPK-OE lines. In contrast, EXOC7-H7-1 was expressed in the 3-dpi and 6-dpi samples from syncytia during the defense response

but not in the 0-dpi samples (Fig. 2). Expression of the exocyst genes that showed expression in the parasitized root cells as well as some of the transgenic MAPK-OE or MAPK-RNAi lines was

confirmed in the transgenic MAPK-OE and MAPK-RNAi lines. Their RNA-seq expression data were confirmed by qRT-PCR using the RPS21 gene as a control. These 4 exocyst genes were EXOC1-1,

EXOC7-B1-1, EXOC7-D1-1 and EXOC7-G1-4 (Fig. 3). During the course of the analysis, a single exocyst gene (EXOC7-H4-1) was found not to be expressed within the syncytium. However, the

increased expression of EXOC7-H4-1 in all 9 of the MAPK-OE lines was confirmed by qRT-PCR using the RPS21 gene as a control (Fig. 3). The observation that EXOC7-H4-1 expression was not

measured within the syncytium but was increased in all 9 of the defense MAPK-OE lines (MAPK-all-OE) is notable. This result indicates that processes involving the _G. max_ secretion

apparatus outside the vicinity of the syncytium are important to defense. This hypothesis was examined later in the analysis. A number of exocyst genes showed increased expression in the

MAPK-OE lines. However, their expression in the parasitized root cells could not be measured by the DCM analysis presented here because probe sets corresponding to those genes were lacking

on the Affymetrix microarray. These genes were EXOC1-2, EXOC3-4, EXOC8-7, EXOC7-C2-2, EXOC7-E2-2, EXOC7-G1-1, EXOC7-G1-3, EXOC7-H4-2, EXOC7-H4-3, EXOC7-H7-2, EXOC7-H7-4 and EXOC7-H7-5

(Supplementary Table S2). The RNA-seq gene expression data revealed that the differential expression of some of these exocyst genes was observed in many of the different transgenic MAPK-OE

lines. The exocyst genes expressed in two or more different transgenic MAPK-OE lines were EXOC1-2 (4 lines), EXOC3-4 (4 lines), EXOC7-C2-2 (5 lines), EXOC7-E2-1 (8 lines), EXOC7-E2-2 (6

lines), EXOC7-H4-2 (2 lines), EXOC7-H4-3 (13 lines), EXOC7-H7-2 (8 lines), and EXOC7-H7-5 (2 lines). In contrast, some of the RNA-seq expression data revealed that the differential

expression of some of these exocyst genes was limited. In these cases, the exocyst gene was expressed in one transgenic MAPK-OE line (Supplementary Table S2). The exocyst genes expressed in

one transgenic line were EXOC1-2, EXOC8-7, EXOC7-G1-1 and EXOC7-H7-4 (Supplementary Table S2). Examination of exocyst genes whose expression was not measured in the parasitized root cells by

DCM (n/a) is beyond the scope of this study (Supplementary Table S2). Consequently, these exocyst genes were not further examined (Supplementary Table S2). Furthermore, examination of the

relationships between other types of signaling processes that may occur and the exocyst is beyond the scope of this analysis. TRANSGENIC PLANTS SHOWED THE EXPECTED EFFECT ON EXOC GENE

EXPRESSION Affymetrix DCM microarray analysis showed that exocyst genes were expressed within parasitized root cells (syncytia) during the defense response. These 16 exocyst genes were

EXOC1-1, EXOC3-5, EXCO4-1, EXOC6-1, EXOC6-6, EXOC7-G1-4, EXOC7-D1-2, EXOC7-E1-1, EXOC5-2, EXOC8-4, EXOC7-B1-2, EXOC7-H7-1, EXOC2-1, EXOC7-A1-3, EXOC7-D1-1 and EXOC7-F1-1. The 16 exocyst

genes were cloned and functionally tested through transgenic analyses. Functional transgenic tests of the exocyst genes were conducted to determine if they play a role in defense. The genes

were overexpressed in the _H. glycines_-susceptible _G. max_[Williams 82/PI 518671] cultivar, after which whether the _H. glycines_-susceptible cultivar became resistant to parasitism was

determined (Fig. 4). In contrast, the same genes were engineered as RNAi cassettes used to decrease their expression in the _H. glycines_-resistant _G. max_[Peking/PI 548402] cultivar. These

functional transgenic tests were conducted to determine whether decreased exocyst gene expression would result in _H. glycines_ susceptibility (Fig. 4). A combination of two outcomes had to

be met for a gene to meet our criteria for a defense role33. First, _H. glycines_ parasitism had to be decreased when the exocyst gene was overexpressed in _H. glycines_-susceptible _G.

max_[Williams 82/PI 518671]. Second, _H. glycines_ parasitism had to be increased when the gene was knocked down by RNAi in _H. glycines_-resistant _G. max_[Peking/PI 548402]. Transgenic

plants in which genes were overexpressed or knocked down via RNAi were shown by expression of the eGFP reporter (Fig. 5). Furthermore, to confirm that the expression cassettes functioned as

expected, qRT-PCR showed that the relative transcript abundance of the exocyst components was increased in the overexpression lines and decreased in the RNAi lines in comparison to that in

the RPS21 gene-expressing control (Fig. 6). The effect of expression of the transgene cassette on root mass was then analyzed. The results demonstrated that expression of the cassettes did

not have a statistically significant effect on root mass when the data were compared to those in the respective controls (_p_ < 0.05) (Fig. 7). However, effects on only root mass were

considered in this analysis. FUNCTIONAL ANALYSIS OF SYNCYTIUM-EXPRESSING EXOCYST GENES DEMONSTRATES A DEFENSE ROLE The effect of altered exocyst gene expression on _H. glycines_ parasitism

was tested. The genetically engineered _G. max_ roots were infected with _H. glycines_ [NL1-Rhg/HG-type 7/race 3] as described in the Materials and Methods section (“Assaying the effect the

genetic engineering events on nematode parasitism” section). The data from the experimental replicates were compared to those from the corresponding controls in the overexpression

(pRAP15-_ccd_B control) and RNAi (pRAP17-_ccd_B control) studies. In the first set of analyses, engineering of the pRAP15-_ccd_B vector in _H. glycines_-susceptible _G. max_[Williams 82/PI

518671] produced a robust level of infection. Quantification of the level of infection showed a cyst count of 203.09 ± 5.04 cysts per wr system and 49.03 ± 5.07 cysts pg of root system. All

exocyst-OE transgenic lines were compared to this standard run in triplicate (please refer to Materials and Methods section “Assaying the effect the genetic engineering events on nematode

parasitism” section for details of the analysis.). In contrast, engineering of the pRAP17-_ccd_B control vector in _H. glycines_-resistant _G. max_[Peking/PI 458402] strongly suppressed

parasitism. Quantification of the level of infection showed 9.94 ± 1.3 cysts per wr system and 2.52 ± 0.43 cysts pg of root system. All exocyst-RNAi transgenic lines were compared to this

standard run in triplicate (please refer to Materials and Methods section “Assaying the effect the genetic engineering events on nematode parasitism” section for details of the analysis.).

The second set of analyses focused on syncytium-expressing exocyst components. Calculation of the FI showed that _H. glycines_ parasitism was significantly reduced by 58–68% in roots

overexpressing each of the exocyst genes in cysts per wr system and by 50–64% in cysts pg of root system (both _p _values < 0.001) (Fig. 8). Consequently, the overexpression of exocyst

components in the _H. glycines_-susceptible _G. max_[Williams 82/PI 518671] cultivar decreased its susceptibility to _H. glycines_ in comparison to that of the respective control. In

contrast, RNAi of the candidate exocyst defense genes in _G. max_[Peking/PI 548402] increased _H. glycines_ parasitism by 3.16–3.77 times in the wr and 4.13–5.68 times pg of root system

(both _p _values < 0.001) (Fig. 8). Consequently, RNAi of the exocyst components increased susceptibility of the _H. glycines_-resistant _G. max_[Peking/PI 458402] cultivar to _H.

glycines_ in comparison to that of the respective control. FUNCTIONAL ANALYSIS OF A MAPK-INDUCED EXOCYST GENES THAT WERE NOT EXPRESSED IN THE SYNCYTIUM The third set of analyses focused on

one exocyst component (EXOC7-H4-1) that was not expressed at any time point within the syncytium during the defense response or in control cells. However, EXOC7-H4-1 expression was confirmed

to be increased in all 9 of the defense MAPK-OE lines by qRT-PCR (Fig. 9). These results indicate that aspects of plant secretion important for the defense process may occur outside of the

parasitized root cells or their progenitors. This observation may explain why higher levels of suppressed _H. glycines_ parasitism were not seen in prior experiments focusing on

syncytium-expressing genes. Alternatively, overexpression of defense MAPKs may synthetically induce the expression of EXOC7-H4-1. In this case, EXOC7-H4-1 may or may not function in defense

at all. To determine whether EXOC7-H4-1 functions in defense, it was cloned and used in overexpression and RNAi experiments. Transgenic EXOC7-H4-1-OE and EXOC7-H4-1-RNAi lines were assessed

by qRT-PCR analyses with RPS21 used as a control, confirming their expected expression (Fig. 10). Analysis of the effect of altered EXOC7-H4-1 transgene cassette expression on root mass was

performed, which demonstrated that the overexpression of EXOC7-H4-1 and RNAi cassette expression did not affect root mass (_p_ > 0.05) (Fig. 11). In replicated functional analyses

employing the same controls used in the previous study, EXOC7-H4-1-OE lines showed _H. glycines_ parasitism that was significantly decreased by 58.8% in the wr and 58.3% pg of root system,

as shown by the FI (_p_ value < 0.001) (Fig. 12). In contrast, _H. glycines_ parasitism in the EXOC7-H4-1-RNAi lines was significantly increased by 3.23-fold in the wr and 4.21-fold pg of

root system, as shown by the FI (_p_ value < 0.001) (Fig. 13). DISCUSSION An analysis of _G. max_ exocyst components is presented here. This study examined whether exocyst components

play a role during the defense response of _G. max_ to the parasitic nematode _H. glycines_. The analysis began by the identification _G. max_ exocyst genes from the most recent Wm82.a2.v1

genome. Then, exocyst genes expressed within the pericycle and surrounding cells prior to _H. glycines_ infection (0 dpi) were determined. Follow-up studies then determined which exocyst

genes are expressed within the syncytium during the defense response to _H. glycines_ infection. The first of the time points selected for analysis was 3 dpi, at which point several

cytological features did not differ between the susceptible and resistant cultivars. The second of the time points selected for analysis was 6 dpi, at which point the cytological features

between the susceptible and resistant cultivars differed, characterizing each reaction. Complimentary analyses were conducted to identify whether the expression of any of the exocyst genes

is under regulation by MAPKs. This analysis is undertaken because studies have demonstrated the importance of MAPKs to the defense response of _G. max_ against _H. glycines_50. These genes

are components of both ETI and PTI50. The subsequent functional, transgenic studies presented here demonstrated that exocyst genes function in defense. The experiments also identified an

exocyst gene that is not expressed within the syncytium during the process of defense but functions in defense. Consequently, the experiments indicated that there processes important to

defense occur both locally within the syncytium and outside of the syncytium. This study began with the identification of all _G. max_ exocyst genes through BLAST searches using the default

parameters in Phytozome with _A. thaliana_ exocyst protein sequences used as queries72. The analysis resulted in the identification of 61 genes that span the 8 exocyst gene families. These

results are consistent with the composition of the exocyst in all eukaryotes, including plants38,40,41,42,43,44,73. The _G. max_ exocyst genes include 5 EXOC1 genes, 2 EXOC2 genes, 5 EXOC3

genes, EXOC4 genes, 2 EXOC5 genes, 6 EXOC6 genes, 31 EXOC7 genes and 8 EXOC8 genes. Consequently, each gene family contains multiple gene copies, which is consistent with the duplicated

nature of the _G. max_ genome74. An analysis of the nature of these gene duplication events is beyond the scope of this study. However, Cvrčková et al_._73 performed phylogenetic analyses of

10 different plant exocyst gene families and obtained important insights into the plant exocyst. The results showed that the small EXOC1, EXOC2, EXOC3, EXOC4 and EXOC5 gene families were

likely amplified independently, late in the diversification of each plant lineage73. Furthermore, the small EXOC6 and EXOC8 gene families were likely amplified from a single ancestral

gene73. In contrast, the very large EXOC7 gene family likely arose from early amplification of an ancestral gene in a common ancestor of land plants73. In each case, gene amplification leads

to the diversification of paralog functions, which require further study. Therefore, the _G. max_ EXOC7 gene family is notably expansive and consists of 31 members. The large size of the

_G. max_ EXOC7 gene family is consistent with observations in other land plants73. _G. max_ exocyst proteins show homology to those in _A. thaliana_ ranging from a low identity of 42.61%

(EXOC7-E2-2) to a high identity of 89.15% (EXOC3-5). However, two outliers in the EXOC8 gene family show a low identity of 60.96% (EXOC8-4) to a high identity of 75.30% (EXOC8-3). These

outliers are EXOC8-6 (25.69% identity) and EXOC8-7 (25.66% identity). It is possible that these proteins are not EXOC8 homologs, and although they were included here, further study is

required. The main objective of this study was to understand whether the _G. max_ exocyst plays a role in defense in the root. Toward that goal, exocyst gene accessions from the most recent

_G. max_ genome assembly (Wm82.a2.v1) were used for the identification of exocyst genes with Affymetrix GeneChip Soybean Genome Array probe set identifiers. This analysis was used to

identify whether any of the exocyst genes exhibit expression specifically within the _H. glycines_-induced syncytium during the defense process. To the best of our knowledge, this is the

only study to sue single-cell transcriptomic analyses from a multicellular organism to specifically identify exocyst genes. The analyses resulted in the identification of 27 of the 61

exocyst genes (44.26%) with an Affymetrix GeneChip Soybean Genome Array probe set identifier. Consequently, it was possible to identify whether any exocyst genes are expressed in the

syncytium during the process of defense. Within that list of 27 exocyst genes, 4 were expressed in the control population of root cells (pericycle and surrounding cells) that were sampled

prior to _H. glycines_ infection (0 dpi). After _H. glycines_ infection, exocyst transcriptomic measurements were performed to detect the expression of 8 genes at 3 dpi during the defense

response. Furthermore, 16 exocyst genes were identified as expressed at the 6-dpi time point during the defense response. The results showed an increase in the number of exocyst genes

expressed during the course of _H. glycines_ parasitism. This expression occurred specifically in _G. max_ root cells during defense reactions (syncytium). Increases in the expression of all

defense genes comprising a large gene family during the course of the defense response were seen in studies of the _G. max_-_H. glycines_ pathosystem34. This study34 examined a family of 22

β-glucosidases predicted to have signal peptides and identified the secreted PEN2 homolog α-hydroxynitrile glucosidase (βg-4), which functions in _G. max_ during its defense response

against _H. glycines_34. The results presented here indicate that the relative transcript abundance of these exocyst components may increase as a consequence of the _G. max_ defense

response. Furthermore, diverse cellular processes involving the _G. max_ exocyst appear to lead to a successful defense response. For example, two different EXOC6 genes (EXOC6-1, EXOC6-6)

and 8 different EXOC7 genes (EXOC7-A1-3, EXOC7-B1-2, EXOC7-D1-1, EXOC7-D1-2, EXOC7-E1-1, EXOC7-F1-1, EXOC7-G1-4 and EXOC7-H7-1) were seen here to function in the defense process (please

refer to “Functional analysis of syncytium-expressing exocyst genes demonstrates a defense role” section: functional analysis of syncytium-expressed exocyst genes). The results indicated

that different types of vesicles containing different cargos may function at specific times or in specific ways during the defense process. Prior analyses have demonstrated the importance of

the MAPK signaling platform to the _G. max_ defense process against _H. glycines_50. MAPKs are part of a central, three-tier signal transduction platform shared by all eukaryotes. MAPKs

permit cells to transduce signals into meaningful output for a variety of physiological and developmental purposes75,76,77. Recently, an analysis of the entire 32-member _G. max_ MAPK gene

family as it relates to defense against _H. glycines_ was conducted50. The analysis led to the identification of 9 MAPKs that function in defense50: MAPK2, MAPK3-1, MAPK3-2, MAPK4-1,

MAPK5-3, MAPK6-2, MAPK13-1, MAPK16-4 and MAPK20-250. In plants, most of the defense processes relating to MAPKs have been shown to involve MAPK3 and MAPK6. Until the work of McNeece et

al_._50, very little evidence regarding the possible defense roles of many of the other MAPKs was available. The observation that _G. max_ MAPKs regulate the expression of exocyst components

that function in defense argues strongly for the broad importance of MAPK signaling in the defense process against _H. glycines_ parasitism. The examination of syncytium-expressing exocyst

genes shown here revealed that a number of these genes were differentially expressed in 1 or more transgenic MAPK lines. For example, EXOC7-B1-1 (an Exo70B1 homolog) had a lower relative

transcript abundance in the MAPK2-RNAi line, in which the _G. max_ defense response against _H. glycines_ was suppressed50. These results are consistent with observations made in _A.

thaliana_ showing that some pathogen effectors impair Exo70B1 protein function through ubiquitination, leading to ETS52. In contrast, the relative transcript abundance of EXOC7-D1-1 was

shown here to be higher in the MAPK3-2-OE line than in the corresponding control line. Furthermore, EXOC7-H7-1, which was expressed in the syncytium at the 3-dpi and 6-dpi time points,

showed higher relative transcript abundance in all of the defense MAPK-OE lines (denoted MAPK-all-OE). The similar expression profile of EXOC7-H4-1 was shown here through its examination in

functional experiments. However, EXOC7-H4-1 was not expressed in syncytial cells analyzed in the Affymetrix DCM study31. These results indicate that secretion processes outside of the

vicinity of the syncytium are important for defense. If signaling processes outside of the syncytium function in defense, systemic processes such as systemic acquired resistance (SAR) may

occur during the _G. max_ defense process against _H. glycines_. _G. max_ genes known to function in SAR, including the transcription factor NONEXPRESSOR of PR1 (NPR1), the lipase ENHANCED

DISEASE SUSCEPTIBILITY 1 (EDS1) and the coiled-coil nucleotide-binding leucine rich repeat (CC-NB-LRR) resistance (R) protein NONRACE SPECIFIC DISEASE RESISTANCE1 (NDR1), have all been shown

to function during the defense against _H. glycines_ parasitism33,50,78,79,80,81,82,83,84. The observation that NDR1 functions in the _G. max_ defense process against _H. glycines_ is

particularly noteworthy since NDR1 is required for ETI. In _A. thaliana_, NDR1 binds three R proteins: the CC-NB-LRR protein RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA1 (RPM1);

RPM1-interacting 4 (RIN4); and the NB adaptor shared by APAF-1, certain _R_ gene products and the CED-4 (ARC)-LRR (NB-ARC-LRR) gene RESISTANT TO P. SYRINGAE 2

(RPS2)79,80,82,85,86,87,88,89,90,91,92. NDR1 induces MAPK gene expression in the _G. max_-_H. glycines_ pathosystem50. Exocyst proteins functioning upstream of vesicle docking act to deliver

callose to infection sites formed by pathogens18,19. This docking process employs the 20 S particle, which incorporates syntaxin (SYP)-containing SNARE. In _A. thaliana_, SYP121 and callose

are delivered to defense sites in plants during resistance to _Botrytis graminis_ f. sp. _hordei_ by the ADP ribosylation factor (ARF)-GTP exchange factor GNOM93. Consistent with those

observations, our prior analyses showed that overexpression of _G. max_ syntaxin 121 resulted in increased callose deposition surrounding the _H. glycines_ syncytium during the defense

response34. In contrast, RNAi of _G. max_ syntaxin 121 decreased callose deposition34. Subsequent follow-up studies identified the expression of 4 different CS genes in the syncytium during

the defense process against _H. glycines_51. Furthermore, overexpression of the different CS genes resulted in a decrease in _H. glycines_ parasitism, while RNAi of those same genes

increased nematode parasitism51. These results provide evidence of processes requiring the exocyst to function in cells that are beyond the boundary of the syncytium during the defense

response. _A. thaliana_ Exo70H4 plays a role in callose deposition in trichomes, consistent with our observations and hypothesis94. The synthetic defense processes due to defense MAPK

overexpression, as it relates to EXOC7-H4-1, are also under examination. Both qRT-PCR and RNA-seq analyses of controls that did not overexpress defense MAPKs detected EXOC7-H4-1 expression.

These results provide evidence that EXOC7-H4-1 expression occurs at least in uninfected tissues that lack syncytia during the defense process. In contrast to these results, the expression of

a number of exocyst genes could not be analyzed by DCM because probe sets for these genes were not included in the Affymetrix GeneChip Soybean Genome Array. However, analysis of their

expression was carried out through the use of data made available by a transgenic MAPK RNA-seq study68. The analysis identified a number of exocysts of genes that were differentially

expressed in one or more transgenic MAPK-OE lines. For example, the EXOC7-E2-1 relative transcript abundance was higher in 8 of the 9 transgenic defense MAPK-OE lines. These _G. max_ exocyst

genes, however, were beyond the scope of the functional analysis presented here because their syncytium expression was not seen (M or NM) by the analytical methods used, referred to as n/a.

These genes will be the focus of future analyses of the _G. max_ exocyst. Infection of genetically engineered exocyst-OE lines with _H. glycines_ resulted in a 58–68% decrease in cysts per

wr system and a 50–64% decrease in cysts pg of root system, depending on the exocyst component under study. Consequently, the _G. max_ overexpression lines exhibited higher susceptibility to

_H. glycines_. In contrast, RNAi studies revealed an increase in _H. glycines_ parasitism of 3.16–3.77-fold in the wr system and a 4.13–5.68-fold increase in the cysts pg of root system

when compared to those in the control. Consequently, the RNAi lines showed an increased susceptibility to _H. glycines_. These results are consistent with observations that

syncytium-expressing genes function in the process of defense32,33,34,37,50,51,83,84. Furthermore, these results are consistent with observations showing that the vesicle transport apparatus

that functions in vesicle docking and membrane fusion also functions in defense32,33,34,37,50,51,83,84. Many of the genes under study in relation to _G. max_ defense against _H. glycines_

showed expression within the syncytium. However, the experiments did not rule out whether gene expression outside of the syncytium is important to the defense process. A recent study

examining the entire _G. max_ MAPK gene family demonstrated that 9 out of 9 MAPKs lacking expression within the syncytium have no role in defense50. These results argue against the functions

of non-syncytium-expressed genes in defense. In contrast, 7 out of 12 syncytium-expressed MAPKs function in defense50. These results demonstrate that most of the MAPKs that function in

defense are expressed within the syncytium. Affymetrix probe sets for the two other MAPKs that function in defense were lacking from the GeneChip Soybean Genome Array, so the syncytium

expression of these MAPKs could not be determined50. Results demonstrating a defense role for genes expressed outside of the vicinity of the syncytium have yet to be determined. The results

presented here reveal that the expression of certain exocyst genes (EXOC7-H4-1) occurs outside of the boundary of the parasitized root cells. In _A. thaliana_, the vesicle transport

machinery involving the exocyst acts at some level to facilitate callose deposition18,19,52,85,95,96,97,98,99,100,101,102. Systemic processes outside of the vicinity of infection as well as

structural modifications of _G. max_ roots, including callose deposition and cell wall modification, have been observed33,34,51,103. These results are consistent with the observation that

these components are coregulated and/or part of a feedback loop that further facilitates the expression of genes that function in the defense process33,34. Over the past few years, a model

of how the process of defense occurs in _G. max_ as it reacts to _H. glycines_ parasitism including the vesicle transport apparatus has been proposed31,32,33,34,37,50,51. This model has its

origins in the demonstration that the 20 S component α-SNAP is specifically expressed within the syncytium during the defense process31. A number of studies have expanded on this theme,

including analyses of the α-SNAP-binding protein syntaxin 31 and the other 20 S particle components, including SNAP-25, synaptobrevin, synaptotagmin, NSF and MUNC1834. These studies

demonstrate the importance of the docking and membrane fusion steps in defense34. These vesicle transport steps are preceded by a tethering process performed by thee exocyst that is

essential for membrane fusion to occur (Fig. 12). Early analyses indicated the importance of vesicle tethering during the _G. max_ defense response to _H. glycines_37. Furthermore, and

earlier analysis demonstrated that _G. max_ Sec4, a protein that is known to function in tethering by binding EXOC6, plays a defensive role against _H. glycines_ parasitism37. Related

experiments have demonstrated that the mechanism by which vesicles are delivered to the cell periphery, which functions through myosin XI, is also important during defense51. These results

all point toward the function of the exocyst at a crucial point in the delivery of vesicles to the site of membrane fusion and plant secretion in the defense process of _G. max_ against _H.

glycines_ parasitism. Furthermore, it appears possible that specific exocyst genes, which are likely the products of duplication events, may function in specific ways to increase the breadth

of the defense response or general health of the plant73. With regard to root-organism interactions, the conserved nature of the exocyst indicates that its function is not limited to the

_G. max_-_H. glycines_ pathosystem, indicating the broad importance of this study. METHODS CANDIDATE GENE SELECTION The Phytozome portal (https://phytozome.jgi.doe.gov) houses the _G. max_

genome sequence and information about its assembly and annotation, making acquisition of the protein sequences of the entire exocyst gene family possible72. _G. max_ exocyst protein

accessions were identified based on comparisons to _A. thaliana_ protein sequences in Phytozome using the Basic Local Alignment Search Tool (BLAST) with the default settings72. These default

settings were as follows: target type: proteome; program: BLASTP (protein query to protein database); expect (E) threshold: − 1; comparison matrix: BLOSUM62; word (W) length: default = 3;

number of alignments to show: 100 allowing for gaps and filter query. Identification and selection of the _G. max_ exocyst defense genes for use in functional transgenic studies were carried

out by using the gene expression data from Matsye et al_._31. These data were obtained through microarray analyses using the GeneChip Soybean Genome Array (Affymetrix). In the study, Matsye

et al_._31 infected two different _G. max_ cultivars that are susceptible or resistant to the _H. glycines_ cultivar under study. _G. max_[Peking/PI 548402] and _G. max_[PI 88788] can be

infected with _H. glycines_[race 14/HG-type 1.3.6.7/TN8], rendering them susceptible. In contrast, _G. max_[Peking/PI 548402] and _G. max_[PI 88788] can be infected with _H.

glycines_[NL1-Rhg/HG-type 7/race 3], rendering them resistant. Laser microdissection (LM) was used to collect pericycle (control) cells prior to _H. glycines_ infection (0 days postinfection

[dpi] control). Syncytia during the resistance process were collected at 3 and 6 dpi. Complementary DNA (cDNA) probes were generated from RNA samples collected at 0, 3 and 6 dpi. The

resulting cDNA probes were used in hybridization experiments with Affymetrix GeneChip Soybean Genome Arrays31,104. These arrays are composed, in part, of 37,744 _G. max_ probe sets covering

35,611 transcripts31,104. The experiments were run in triplicate31,104. This experimental process resulted in the production of 6 total arrays for each time point (_G. max_[Peking/PI

548402]: arrays 1–3; _G. max_[PI 88788]: arrays 1–3), which were used to determine whether the presence of transcript corresponding to the probe set was demonstrably different from zero

(present [P]), uncertain (marginal [G]), or not demonstrably different from zero (absent [A])31,104. For our purposes, a gene was considered to be measured [M] when the probe signal was

detectable above a threshold (_p_ < 0.05) on all 6 arrays (the 3 arrays each from _G. max_[Peking/PI 548402] and _G. max_[PI 88788])31,104. For the analysis presented here, the expression

of an exocyst gene was considered not measured (NM) if the probe signal was not detected at a statistically significant level (_p_ ≥ 0.05) on any of the 6 arrays. For some genes, no

corresponding probe set was fabricated onto the microarray. In these cases, gene expression was not determined and considered not applicable (n/a). For this part of the analysis, the

Affymetrix annotations were mapped to the original _G. max_ genome release (Wm82_._a1_._v1_._1) since only that annotation was available at the time of the analysis31. Here, these older

annotations are compared to the updated, most recent _G. max_ Wm82.a2.v1 genome assembly and annotation. RNA SEQUENCING RNA sequencing (RNA-seq) data were obtained from the experiments of

McNeece et al_._50 and Alshehri et al_._68. These studies examined the _G. max_ MAPK gene family as it relates to the defense of _G. max_ against _H. glycines_ parasitism and showed that 9

of the 32 _G. max_ MAPKs function during the defense reaction against _H. glycines_ parasitism, naming these MAPKs defense MAPKs50. Single replicate RNA-seq experiments examining the 9

defense MAPKs were performed using RNA isolated from transgenic lines in which the targeted MAPK genes were either overexpressed (OE) or inhibited via RNAi50,68. The defense MAPKs examined

were MAPK2 (Glyma.06G029700), MAPK3-1 (Glyma.U021800), MAPK 3-2 (Glyma.12G073000), MAPK 4-1 (Glyma.07G066800), MAPK 5-3 (Glyma.08G017400), MAPK6-2 (Glyma.02G138800), MAPK 13-1

(Glyma.12G073700), MAPK16-4 (Glyma07g38510) and MAPK20-2 (Glyma.14G028100), and pRAP15-_ccd_B and pRAP17-_ccd_B served as corresponding controls50,68. RNA was isolated from the 9 defense

MAPK-OE and MAPK-RNAi lines and their respective controls, and the RNA sequences were deposited and made publicly available68. For the experimental purposes presented here, an additional

goal was the identification of exocyst genes whose expression was induced or suppressed by the different studied MAPKs. Expression of the exocyst genes that met the differential expression

criterion in the RNA-seq experiments (± 1.5-fold change in expression, _p_ < 0.05) was confirmed by quantitative real-time PCR (qRT-PCR) as described in the Materials and Methods section

(“cDNA synthesis” and “qRT-PCR assessment of gene expression” sections). Expression of the remaining exocyst genes that did not meet the differential expression criterion in the RNA-seq

analyses (NDE) was not confirmed by qRT-PCR. The _G. max_ genome accessions were used to mine exocyst RNA-seq gene expression data from the defense MAPK RNA-seq study and are shown in the

analysis50,68. The _G. max_ accession numbers whose RNA-seq data are presented were derived from the most recent _Glycine max_ Wm82.a2.v1 annotation. The exocyst accession numbers were

further manually confirmed with Phytozome to confirm their accuracy (as of February 15, 2020)72,74. FUNCTIONAL TESTING OF CANDIDATE DEFENSE GENES The candidate _G. max_ exocyst defense gene

sequences were extracted from Phytozome72,74, cloned and overexpressed in the _H. glycines_-susceptible cultivar _G. max_[Williams 82/PI 518671]50. In addition, the candidate exocyst defense

genes were cloned for RNAi in the _H. glycines_-resistant cultivar _G. max_[Peking/PI 548402]50. PCR primer sequences were developed against the exocyst component transcript sequences from

the recent _Glycine max_ Wm82.a2.v1 annotation and confirmed to match the Wm82.a2.v1 genome (February 15, 2020) (Supplementary Table S3). Candidate _G. max_ exocyst defense gene amplicons

were synthesized by PCR using the AccuPrime Taq Polymerase System (Invitrogen) according to the manufacturer’s instructions with an Eppendorf AG Mastercycler Pro S model 6,325 PCR gradient

PCR thermocycler. The reaction conditions were dependent on the nucleotide composition of the amplicon and PCR primer. In general, DNA melting was carried out at 95 °C for 2 min, followed by

another 30-s melt at 95 °C. Primer annealing conditions were empirically determined through gradient PCR for 30 s. Primer extension was carried out at 68 °C for 1 min per 1,000 base pairs

of the sequence. This process was carried out for 35 cycles, followed by a final step at 68 °C for 10 min, with the reaction completed at 4 °C. The PCR product was run on a 1% agarose gel.

The amplicons were removed from the gel and purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s instructions. Subsequently, the amplicon was

ligated into the pENTR/D-TOPO entry vector using the pENTR/D-TOPO Cloning Kit (Invitrogen) according to the manufacturer’s instructions. The reaction contents were transformed into One Shot

TOP10 chemically competent _E. coli_ (TOP 10) (Invitrogen) cells according to the manufacturer’s instructions as described. Cells were selected on Luria–Bertani (LB) agar plates containing

50 μg/ml kanamycin. Plasmid DNA was isolated from selected colonies using the Wizard _Plus_ SV Minipreps DNA Purification System (Promega) according to the manufacturer’s instructions. The

DNA sequences were confirmed by Sanger sequencing. Subsequently, the exocyst amplicons were ligated to Gateway-compatible overexpression (pRAP15) or RNAi (pRAP17) destination vectors using

Gateway LR Clonase Enzyme Mix (Invitrogen) according to their instructions to transfer the candidate _G. max_ exocyst resistance gene amplicon into the respective destination vectors.

Nonengineered pRAP15 and pRAP17 vectors served as experimental controls; these vectors contained the _ccd_B gene where the candidate _G. max_ exocyst defense gene amplicon would otherwise be

following directional insertion during the LR clonase reaction. Based on this feature, the nonengineered pRAP15-_ccd_B (overexpression control) and pRAP17-_ccd_B (RNAi control) vectors were

suitable controls to account for any nonspecific effects of gene overexpression or RNAi50. The reaction contents were transformed into chemically competent _E. coli_ TOP 10 cells according

to the manufacturer’s instructions as described. Cells were selected on LB agar plates containing 5 μg/ml tetracycline. _E. coli_ colonies containing the gene of interest (GOI) after

transformation with pRAP15/17-GOI were grown in 3 ml of liquid LB medium and chemically selected with 5 μg/ml tetracycline overnight at 37 °C. Plasmid preps (Promega) of these liquid

cultures were carried out according to the manufacturer’s instructions. Gene-specific primers were used to confirm the presence of each exocyst gene (Supplementary Table S3). The pRAP15/17

destination vectors confirmed to have the exocyst gene amplicon were transformed into chemically competent _Agrobacterium rhizogenes_ K599 (K599)50 via freeze–thaw transformation50. In this

procedure, 250 μl of K599 cells was thawed on ice. A sufficient amount of plasmid DNA (0.1–1 μg) was added to K599 cells and gently mixed. The mixture of K599 cells and plasmid DNA was

incubated on ice for 5 min and then subsequently transferred to liquid N2 for 5 min. The mixture was transferred to a 37 °C water bath for 5 min. The contents were then transferred to a

culture tube with 1 ml of LB medium, placed in a shaking incubator, and incubated at 28 °C for 2 h. The cells were then collected by centrifugation for 2 min at 5,000 rpm, resuspended in 200

 μl of LB medium and spread on LB agar plates containing 5 μg/ml tetracycline for chemical selection at 28 °C. After 2 days, K599 colonies that underwent genetic transformation were picked

for confirmation of the DNA cassette with _G. max_ exocyst gene-specific primers. Colonies harboring the appropriate plasmid were then grown in 250 ml of LB medium containing 5 μg/ml

tetracycline at 28 °C in a shaking incubator50. PRODUCTION OF TRANSGENIC PLANTS FOR FUNCTIONAL EXPERIMENTS A solution of K599 cells transformed with the appropriate vector construct was

pelleted by centrifugation in a Sorvall RC6 Plus Superspeed Centrifuge at 4 °C for 20 min. The resulting pellet of K599 cells was resuspended in Murashige and Skoog medium containing

vitamins (MS) (Duchefa Biochemie) and 3.0% sucrose at pH 5.7 (MS medium)105. Transgenic _G. max_ production began when the root of each 1-week-old plant was cut off at the hypocotyl with a

new, sterile razor blade that had been immersed in the K599 cell solution in a Petri dish. This procedure allowed the transformed K599 cells access to the wound made by removal of the root.

Subsequently, 25 root-less plants were placed in a 140-ml glass beaker with 25 ml of transformed K599 cells in MS medium. The plants were placed under vacuum using the VP60 Two Stage Vacuum

Pump (CPS Products, Inc.) in a Bel-Art “Space Saver” polycarbonate vacuum desiccator with a clear polycarbonate bottom (Bel-Art) for 5 min. The plants were then left under vacuum for 10 min.

The vacuum was then slowly released, allowing the transformed K599 cells to further enter the plant tissue. After cocultivation, the cut ends of _G. max_ were individually placed 3–4 cm

deep into fresh coarse grade A-3 vermiculite (Palmetto Vermiculite) in 50-cell propagation trays (725602C) held in standard flats (710245C) with holes in the bottom (T.O. Plastics). The

plant trays are were placed in sterilize 25-qt./23-L modular latched boxes (Sterlite) that were then covered with their lids. The covered modular latched boxes were placed at a distance of

20 cm from standard fluorescent cool white 4,100-K/32-W bulbs emitting 2,800 lumens (Sylvania) for 5 days at ambient laboratory temperature (22 °C). The plants were transferred to the

greenhouse and removed from the modular latched boxes for recovery for 1 week. Visual selection of transgenic _G. max_ roots was carried out with the enhanced green fluorescent protein

(eGFP) reporter using a Dark Reader Spot Lamp (SL10S) (Clare Chemical Research)50. Roots exhibiting eGFP reporter expression also possessed the candidate defense gene expression cassette,

and each had its own promoter and terminator sequences. Gene transfer occurred because K599 cells transported the DNA cassettes between the left and right borders of the pRAP15 and pRAP17

destination vectors into the root cell chromosomal DNA. The result was a stable transformation event in the root somatic cell, even though the DNA cassette had not been incorporated into the

germline. Roots subsequently developed from the transgenic cell over a period of a few weeks. The resultant genetically mosaic plants had a nontransgenic shoot with a transgenic root

system. Therefore, in the experiments presented here, each individual transgenic root system is an independent transformant line. The transgenic plants were each planted in a Ray Leach

“Cone-tainer” (SC10) (Stuewe and Sons, Inc.) secured in a Ray Leach Tray (RL98) (Stuewe and Sons, Inc.) in sandy (93.00% sand, 5.75% silt, and 1.25% clay) soil and allowed to recover for two

weeks prior to the start of the experiment50. The functionality of the genetic constructs in _G. max_ was confirmed by qRT-PCR (please refer to the quantitative real-time PCR

[qRT-PCR]-related Sects. 4.5 and 4.6). CDNA SYNTHESIS _G. max_ root RNA was isolated using the UltraClean Plant RNA Isolation Kit (Mo Bio Laboratories, Inc.) according to the manufacturer’s

instructions50. The RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) according to the manufacturer’s instructions. Genomic DNA was removed from the RNA

with amplification-grade DNase I (Invitrogen) according to the manufacturer’s instructions. With oligo d(t) used as the primer, the SuperScript First Strand Synthesis System for RT-PCR

(Invitrogen) was used to synthesize cDNA from mRNA according to the manufacturer’s instructions. Genomic DNA contamination was assessed using a cupin (Glyma.20G148300) primer pair that

amplifies DNA across an intron (Supplementary Table S3)32. The PCR experiments yielded differently sized products based on the presence or absence of that intron32. QRT-PCR ASSESSMENT OF

GENE EXPRESSION Candidate exocyst defense gene expression in transgenic _G. max_ was assessed by qRT-PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems), TaqMan

6-carboxyfluorescein (6-FAM)-labeled probes and Black Hole Quencher-1 (BHQ1) (MWG Operon) (Supplementary Table S3) according to the manufacturer’s instructions50. A qRT-PCR control designed

from a ribosomal S21 (RPS21) protein-coding gene (Glyma.15G147700) was used in the _G. max_ experiments (Supplementary Table S3)50. Fold changes in gene expression caused by the genetic

engineering events were calculated using the 2−ΔΔ_CT_ method50,69. Student’s _t_-test was used to calculate the _p_ values for the replicate qRT-PCR experiments70. The procedures followed

those presented by McNeece et al_._50. ASSAYING THE EFFECT THE GENETIC ENGINEERING EVENTS ON NEMATODE PARASITISM Infection of the transgenic plants with _H. glycines_ was performed according

to the procedures described by Sharma et al_._34. _H. glycines_ eggs were obtained from cysts collected from 60-day-old, greenhouse-grown _G. max_ stock cultures. The cultures were

maintained in 500-cm3 polystyrene pots. Stock _H. glycines_ cysts were purified by sucrose flotation106. _G. max_ roots that contained _H. glycines_ cysts were washed through nested sieves

with pore sizes of 850 μm and 250 μm. The _H. glycines_ cysts were collected from the 250-μm sieve after this procedure and ground with a mortar and pestle to release the eggs. The _H.

glycines_ eggs were obtained after gravitational sieving followed by sucrose centrifugation. The _H. glycines_ eggs were recovered with a 75-μm sieve nested over a 25-μm sieve. _H. glycines_

J2s were collected from hatched eggs in a modified Baermann funnel placed on a Slide Warmer (model 77) (Marshall Scientific) at 28 °C. _H. glycines_ eggs hatched from days 4 to 7. _H.

glycines_ J2s were collected on a sieve with a 25-μm pore sire and placed in 1.5-ml tubes. The tube and its contents were centrifuged at 10,000 rpm for 1 min, washed with distilled sterile

water and centrifuged again. The J2s were concentrated by centrifugation in an IEC clinical centrifuge for 30 s at 1,720 rpm to a final optimized concentration of 2,000 pi-J2/ml. Each root

was inoculated with one ml of _H. glycines_ at a concentration of 2,000 J2s/ml per root system (per plant). Infection was allowed to proceed for 30 days. At the end of the experiment, the

cysts were collected over nested 20- and 100-mesh sieves34. Furthermore, the soil was washed several times, and the rinse water was sieved to assure collection of all cysts for enumeration

of the female index (FI)34. The FI, the community-accepted standard representation of the obtained data24, was calculated by a procedure originally described by Golden et al_._24 as follows

and employed for functional transgenic experiments50: FI = (Nx/Ns) × 100. In the procedure employed here, Nx was the pRAP-exocyst gene-transformed (experimental) line. Ns was the pRAP-_ccd_B

(control) line34. The FI was calculated as the number of cysts per whole root (wr) system grown within 100 cc of soil and the number of cysts per gram (pg) of root system50. Historically,

analysis by wr system has been the method of choice for data presentation24. Analysis of the number of cysts pg of root system, however, accounts for possible changes in root growth caused

by the influence of the overexpression or RNAi of the candidate _G. max_ exocyst defense gene. Three biological replicates consisting of 10–20 individual transgenic plants each were made for

each construct. The results were statistically examined using the Mann–Whitney–Wilcoxon (MWW) rank-sum test, a nonparametric test of the null hypothesis that does not require the assumption

of a normal distribution, with a cutoff of _p_ < 0.0550,71. DATA AVAILABILITY Data relevant to the study is presented here as supplemental data. ABBREVIATIONS * DCM: Detection call

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ACKNOWLEDGEMENTS The authors are thankful for start-up support and teaching assistantships provided by the Department of Biological Sciences, Mississippi State University. In addition, the

authors are thankful for an awarded competitive Special Research Initiative grant from the College of Arts and Sciences at Mississippi State University (VK), which funded the RNA-seq

experiments. The authors are also thankful to the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University for greenhouse space.

Furthermore, the authors are thankful to Gary Lawrence, formerly of the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, for his

support throughout these studies. The authors thank Sudha Acharya, a graduate student in the Department of Biological Sciences, for her contributions to the study. The authors would like to

thank a large contingent of undergraduate students who contributed in important ways to these studies over the years. These students include Christina Jones, Dollie Welch, Priyanka Gadre,

Katherine Thrash, Adrienne McMorris, Chase Robinson, Danielle Francis, Brittany Ginn, Kara Jackson, Suchit Salian, Olivia Long, Hannah Burson, Meghan Calhoun, Nishi Sunthwal, John Clune,

Taylor Henry, Madison Milhoan, Kayla Moore, Neil Shannon, Ashley Dowdy, Katherine McCracken, Erin Curran, Annedrea McMillan, Austin Martindale, Keigero Fergusen, Alison Antee, Hannah Miller,

Tineka Burkhead, Henry Pittman, Erin Ball, Jamelle Vance, Leslie Canale, Courtney Gagliano, Shelby Janeski, Lauren Langston, Eileen Modzeleski, Natalie Rentrop, Hannah Austin, Carolyn

Chacon, Emily Carter, Erica Sowell, Jody Clark, Chelsea Tittle, Chrissy Miller, Hannah Stimson, Kathryn Stiglet, Ashlee Vargason, Robyn Beattie, Anna Bailey Britt, Harshini Sampathkumar,

Henderson Rand, Aishwarya Dikshit, Dejanie Dilworth, Samantha Rushing, Kyle Winston, Meagan Young, Morgan Urich, Chase Nash, Makenzie Miller, Rebecca Waters, Anna-Marie Autrey, Maggie Kuhn,

Kelvin Blade, Jesse Austin, Haleigh Smith, Alex Hammett, Caroline Knesal, Landon Heineck, Caleb Stallings, Santana Holloway, Candace Wyatt, Caroline Hoggard, Cassady Knudsen, Mathilda Lail,

Ellen Condoure, Morgan Castaneda, Rebecca Billingsley, Seth Lenoir, Suraj Neupane, Jared Quave, Swechchha Tamang, Jilanna Simmons, Lukas Wicht, Emily Sosnowski, Courtney Borgognoni, Rashada

Boler, Hannah Cox, Madison Baima, Murry Faulkner, Gill Goodloe, Sarah Heifner, Holeh Heydari, Drake Pace, Cameron Roach, Kim Anderson, Ngoc Pham, Ana Simal, Adam Crittenden, Anna Gaudin,

Cassady Knudsen, and Cheyenne Golden. The authors are very thankful to the Shackouls Honors College. FUNDING Funding was provided by Mississippi State University, College of Arts and

Sciences Grant No. (CAS 2018). AUTHOR INFORMATION Author notes * Keshav Sharma Present address: USDA-ARS Cereal Disease Laboratory, University of Minnesota, 1551 Lindig Street, St. Paul, MN,

55108, USA * Prakash M. Niraula Present address: Department of Plant Pathology and Microbiology, Texas A&M AgriLife Research and Extension Center, Texas A&M University, 2415 E. Hwy.

83, Weslaco, TX, 78596, USA AUTHORS AND AFFILIATIONS * Department of Biological Sciences, Mississippi State University, Mississippi State, MS, 39762, USA Keshav Sharma, Prakash M. Niraula, 

Hallie A. Troell, Mandeep Adhikari & Vincent P. Klink * Department of Mathematics and Computer Science, Texas Women’s University, Denton, TX, 76204, USA Hamdan Ali Alshehri * Department

of Computer and Information Sciences, Towson University, Towson, MD, 21252, USA Nadim W. Alkharouf * Department of Entomology and Plant Pathology, Auburn University, 209 Life Science

Building, Auburn, AL, 36849, USA Kathy S. Lawrence * Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS,

39762, USA Vincent P. Klink * Center for Computational Sciences High Performance Computing Collaboratory, Mississippi State University, Mississippi State, MS, 39762, USA Vincent P. Klink

Authors * Keshav Sharma View author publications You can also search for this author inPubMed Google Scholar * Prakash M. Niraula View author publications You can also search for this author

inPubMed Google Scholar * Hallie A. Troell View author publications You can also search for this author inPubMed Google Scholar * Mandeep Adhikari View author publications You can also

search for this author inPubMed Google Scholar * Hamdan Ali Alshehri View author publications You can also search for this author inPubMed Google Scholar * Nadim W. Alkharouf View author

publications You can also search for this author inPubMed Google Scholar * Kathy S. Lawrence View author publications You can also search for this author inPubMed Google Scholar * Vincent P.

Klink View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.S. was involved in the cloning, transgenic work, generating the transgenic lines,

qRT-PCR, data collection, and data interpretation and produced the manuscript. P.M.N. was involved in generating the transgenic lines, data collection and interpretation of results. H.A.T.

conducted analyses involving the RNA-seq and DCM data. M.A. conducted analyses involving the RNA-seq and DCM data. H.A.A. was involved in the RNA-seq analyses. N.W.A. was involved in the

RNA-seq analyses and overseeing those analyses. K.S.L. was involved in the nematode analyses. V.P.K. designed the experiments, generated RNA-seq and qRT-PCR data, performed analyses with

prior gene expression data, interpreted the results and produced the manuscript. CORRESPONDING AUTHOR Correspondence to Vincent P. Klink. ETHICS DECLARATIONS COMPETING INTERESTS The authors

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visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Sharma, K., Niraula, P.M., Troell, H.A. _et al._ Exocyst components promote

an incompatible interaction between _Glycine max_ (soybean) and _Heterodera glycines_ (the soybean cyst nematode). _Sci Rep_ 10, 15003 (2020). https://doi.org/10.1038/s41598-020-72126-z

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