Targeted suppression of soybean BAG6-induced cell death in yeast by soybean cyst nematode effectors

While numerous effectors that suppress plant immunity have been identified from bacteria, fungi, and oomycete pathogens, relatively little is known for nematode effectors. Several dozen effectors have been reported from the soybean cyst nematode (SCN). Previous studies suggest that a hypersensitive response-like programmed cell death is triggered at nematode feeding sites in soybean during an incompatible interaction. However, virulent SCN populations overcome this incompatibility using unknown mechanisms. A soybean BAG6 (Bcl-2 associated anthanogene 6) gene previously reported by us to be highly up-regulated in degenerating feeding sites induced by SCN in a resistant soybean line was attenuated in response to a virulent SCN population. We show that GmBAG6-1 induces cell death in yeast like its Arabidopsis homolog AtBAG6 and also in soybean. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean defence responses during infection. Thus, we used a yeast viability assay to screen SCN effector candidates for their ability to specifically suppress GmBAG6-1-induced cell death. We identified several effectors that strongly suppressed cell death mediated by GmBAG6-1. Two effectors identified as suppressors showed direct interaction with GmBAG6-1 in yeast, suggesting that one mechanism of cell death suppression may occur through an interaction with this host protein.     

Transgenic soybean overexpressing GmSAMT1 exhibits resistance to multiple‐HG types of soybean cyst nematode Heterodera glycines

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCN resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1‐year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Thus, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production.     

A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73728 clones stored in 192384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.     

A single region of the Phytophthora infestans avirulence effector Avr3b functions in both cell death induction and plant immunity suppression

As a destructive plant pathogen, Phytophthora infestans secretes diverse host-entering RxLR effectors to facilitate infection. One critical RxLR effector, PiAvr3b, not only induces effector-triggered immunity (ETI), which is associated with the potato resistance protein StR3b, but also suppresses pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). To date, the molecular basis underlying such dual activities remains unknown. Based on phylogenetic analysis of global P. infestans isolates, we found two PiAvr3b isoforms that differ by three amino acids. Despite this sequence variation, the two isoforms retain the same properties in activating the StR3b-mediated hypersensitive response (HR) and inhibiting necrosis induced by three PAMPs (PiNpp, PiINF1, and PsXeg1) and an RxLR effector (Pi10232). Using a combined mutagenesis approach, we found that the dual activities of PiAvr3b were tightly linked and determined by 88 amino acids at the C-terminus. We further determined that either the W60 or the E134 residue of PiAvr3b was essential for triggering StR3b-associated HR and inhibiting PiNpp- and Pi10232-associated necrosis, while the S99 residue partially contributed to PTI suppression. Additionally, nuclear localization of PiAvr3b was required to stimulate HR and suppress PTI, but not to inhibit Pi10232-associated cell death. Our study revealed that PiAvr3b suppresses the plant immune response at different subcellular locations and provides an example in which a single amino acid of an RxLR effector links ETI induction and cell death suppression.     

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection.     

Loci underlying resistance to Race 3 of soybean cyst nematode in Glycine soja plant introduction 468916

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is an important soybean [Glycine max (L.) Merr.] pest in the U.S. and throughout the world. Genetic resistance is the primary method for controlling SCN and there is a need to identify new resistance genes. Glycine soja Sieb. and Zucc. is the wild ancestor of domesticated soybean and is a potential source of new SCN resistance genes. The goal of this research was to map quantitative trait loci (QTLs) that provide resistance to SCN Race 3 from the G. soja plant introduction (PI) 468916. Fifty seven F₂-derived lines from a cross between the G. soja PI 468916 and the G. max experimental line A81-356022 were tested for resistance to an SCN population with a Race-3 phenotype. These lines were also genotyped with 1,004 genetic markers and resistance genes were mapped by composite interval mapping with the computer program QTL-Cartographer. In the F₂ population, three significant (LOD > 3.0) QTLs were detected that explained from 5% to 27% of the variation for Race-3 resistance. The two most significant QTLs identified in the F₂ population were tested in a population of 100 BC1F₂ plants developed by crossing A81-356022 to a line from the F₂ population that carried the two resistance QTLs from G. soja. In the backcross population, both Race-3 resistance QTLs were significant, which confirms the existence of these QTLs. The QTLs identified in this experiment map to positions where SCN resistance genes have not been previously identified, suggesting that these are novel genes that could be useful for diversifying the resistance genes currently used in cultivar development. Received: 7 August 2000 / Accepted: 4 December 2000     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.     

A Meloidogyne graminicola C-type lectin,Mg01965, is secreted into the host apoplast to suppress plant defence and promote parasitism

C-type lectins (CTLs), a class of multifunctional proteins, are numerous in nematodes. One CTL gene, Mg01965, shown to be expressed in the subventral glands, especially in the second-stage juveniles of the root-knot nematode Meloidogyne graminicola, was further analysed in this study. In vitro RNA interference targeting Mg01965 in the preparasitic juveniles significantly reduced their ability to infect host plant roots. Immunolocalizations showed that Mg01965 is secreted by M. graminicola into the roots during the early parasitic stages and accumulates in the apoplast. Transient expression of Mg01965 in Nicotiana benthamiana and targeting it to the apoplast suppressed the burst of reactive oxygen species triggered by flg22. The CTL Mg01965 suppresses plant innate immunity in the host apoplast, promoting nematode parasitism in the early infection stages.     

Linkage of soybean cyst nematode alleles for incompatibility with soybean

Selection and inbreeding of soybean cyst nematodes increased populations' ability to produce cysts on some soybean lines with concurrent decreases in numbers of cysts on other soybean lines: evidence that some alleles for incompatibility were either linked or at the same loci. Some responses could be explained only by linkage of nematode genes for avirulence. Linkage of nematode alleles for incompatibility could be involved when selection increased numbers of cysts on several lines even though the usual interpretation has been that the lines had some of the same genes for resistance. Most of the lines used in this study may have fewer alleles for incompatibility than most "resistant" lines. Use of these lines with fewer genes for resistance should help in the identification of individual alleles for incompatibility necessary for resolving the allelism and/or linkage of these nematode genes.     

Interactions between iron-deficiency chlorosis and soybean cyst nematode in Minnesota soybean fields

Experiments were conducted in four commercial fields differing in severity of iron-deficiency chlorosis (IDC), and soybean cyst nematode (SCN) in Waseca and Lamberton, Minnesota to determine the interaction between the IDC and SCN. Each experiment was a randomized complete block with a factorial treatment design including 23 cultivars with or without traits of resistance to SCN, and IDC. The study illustrated the interactive effects of the two defensive traits on the diseases and soybean yields. IDC rating was higher in SCN-susceptible than SCN-resistant soybean, suggesting SCN infection increased IDC. Resistance to IDC apparently increased SCN reproduction due to better soybean plant growth. Yield response to the defensive traits depended on the disease pressures in a field. When both IDC and SCN were present in a field, deploying SCN-resistance was the best solution to the problems. However, SCN-resistance suppressed soybean yields when used in fields without the disease problems. IDC-resistance increased yield of SCN-susceptible cultivars, but it did not result in detectable yield benefit of SCN-resistant cultivars in SCN-infested sites. Effective use of the defensive traits for management of IDC and SCN requires specific knowledge of the disease problems present in a field. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture and the University of Minnesota.     

Molecular markers for resistance to Heterodera glycines in advanced soybean germplasm

Germplasm line J87-233 is resistant to soybean cyst nematode (SCN) races 1, 2, 3, 5 and moderately resistant to race 14 with resistance derived from 3 primitive sources, Peking, PI 88788 and PI 90763. F_2:3 progeny of J87-233 and SCN-susceptible Hutcheson cross were evaluated for response to SCN races 1, 2, 3, 5 and 14. Linkage groups (LG) A, B, F, G, J, M, N, S were tested with 215 genomic clones and 45 decamers for parental genotypes. QTL for race 1 and QTL for race 3 were detected on LG A2, the region of BLT65V and SCAR 548/563_{1100/1025,975.} The cluster analysis of 12 soybean cultivars and 38 plant introductions confirmed association of SCAR_{1100/1025,975} with resistance to races 1 and 3, and suggested possible DNA rearrangements that might give rise to new resistance specificities in the region. The highly significant association of K69T marker with SCN race 1 resistance in conjunction with its location, 18.5 cM from the reported QTL, exemplifies the importance of the QTL locus on LG G and suggests expansion of the linkage map in the LG G-terminal region. Detected interaction between loci on LG A2 and LG G, and also with loci on LG F and LG M, may play a significant role in the genotype-specific response to SCN. Identification of two major regions on LG A2 and LG G for SCN resistance shows their applicability to advanced germplasm, however, transmission of molecular marker alleles indicates that applied markers are not yet reliable in revealing all possible recombination events in breeding for SCN resistance.     

SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance

Background

Soybean cyst nematode (SCN) is the most economically devastating pathogen of soybean. Two resistance loci, Rhg1 and Rhg4 primarily contribute resistance to SCN race 3 in soybean. Peking and PI 88788 are the two major sources of SCN resistance with Peking requiring both Rhg1 and Rhg4 alleles and PI 88788 only the Rhg1 allele. Although simple sequence repeat (SSR) markers have been reported for both loci, they are linked markers and limited to be applied in breeding programs due to accuracy, throughput and cost of detection methods. The objectives of this study were to develop robust functional marker assays for high-throughput selection of SCN resistance and to differentiate the sources of resistance.

Results

Based on the genomic DNA sequences of 27 soybean lines with known SCN phenotypes, we have developed Kompetitive Allele Specific PCR (KASP) assays for two Single nucleotide polymorphisms (SNPs) from Glyma08g11490 for the selection of the Rhg4 resistance allele. Moreover, the genomic DNA of Glyma18g02590 at the Rhg1 locus from 11 soybean lines and cDNA of Forrest, Essex, Williams 82 and PI 88788 were fully sequenced. Pairwise sequence alignment revealed seven SNPs/insertion/deletions (InDels), five in the 6th exon and two in the last exon. Using the same 27 soybean lines, we identified one SNP that can be used to select the Rhg1 resistance allele and another SNP that can be employed to differentiate Peking and PI 88788-type resistance. These SNP markers have been validated and a strong correlation was observed between the SNP genotypes and reactions to SCN race 3 using a panel of 153 soybean lines, as well as a bi-parental population, F₅–derived recombinant inbred lines (RILs) from G00-3213 x LG04-6000.

Conclusions

Three functional SNP markers (two for Rhg1 locus and one for Rhg4 locus) were identified that could provide genotype information for the selection of SCN resistance and differentiate Peking from PI 88788 source for most germplasm lines. The robust KASP SNP marker assays were developed. In most contexts, use of one or two of these markers is sufficient for high-throughput marker-assisted selection of plants that will exhibit SCN resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1531-3) contains supplementary material, which is available to authorized users.     

The molecular dialog between oomycete effectors and their plant and animal hosts

Oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that include some of the most notorious pathogens of plants and animals. Through the deployment of a remarkably diverse array of effector proteins, oomycete pathogens succeed to overcome host defences and cause infection. Effectors can operate extracellularly or enter living cells where they target diverse subcellular compartments. Genome sequence information indicates that oomycetes express several hundred host-translocating effectors potentially targeting a myriad of host processes. To counteract, plants rely on a wide variety of extra- and intracellular immune receptors facilitating pattern-triggered and effector-triggered immunity, respectively. Similarly, effectors from animal pathogenic oomycetes also target host immune response pathways, which in turn causes the activation of the humoral and adaptive immune system. In this review, we compare plant and animal pathogenic oomycete effectors regarding their type, function, genetic diversity, as well as host responses.     

Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

 The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg ₄ locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg ₄ allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg ₄ ) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg ₄) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj ₄ ). A segregating population of 80 F₂ and F₃ plants derived from the cross PI 290136×BARC-2 (Rj ₄ ) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg ₄ locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved. Received: 8 January 1998 / Accepted: 15 July 1998     

Association of RFLP markers with loci conferring broad-based resistance to the soybean cyst nematode (Heterodera glycines)

Soybean cyst nematode (SCN) is a major soybean yield-limiting pest. The present study was conducted to map broad-based SCN resistance loci from the cultivar Hartwig. Two-hundred F₂₃ lines derived from the cross Williams 82 x Hartwig were screened with a fourth-generation SCN inbred and 56 polymorphic molecular markers. Allele states and phenotypes were analyzed using stepwise regression and the model selection was made at P 0.01. Four unlinked RFLP markers (A006, A567, A487, A112) were associated with SCN resistance and the partial coefficient of determinations (R²) were 91%, 1%, 1%, and 1%. We have mapped a new, major SCN resistance locus (A006) and three minor loci (A567, A487, A112). This complete mapping will accelerate the transfer of broad-based resistance without linkage drag and aid in the determination of relationships among various SCN-resistant germplasm sources.     

Two simple sequence repeat markers to select for soybean cyst nematode resistance coditioned by the rhg1 locus

The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the most economically significant soybean pest. The principal strategy to reduce or eliminate damage from this pest is the use of resistant cultivars. Identifying resistant segregants in a breeding program is a difficult and expensive process which is complicated by the oligogenic nature of the resistance and genetic variability in the pathogen. Fortunately, resistance at one SCN-resistance locus, rhg1, is generally accepted as a necessity for the development of resistant genotypes using any source of resistance and when challenged by any SCN race. Thus, the development of SCN resistant cultivars would be expedited if an effective and rapid system were available to identify breeding lines carrying a resistance allele at the rhg1 locus. In this study we report two simple sequence repeat (SSR) or microsatellite loci that cosegregate and map 0.4 cM from rhg1. Allelic variation at the first of these loci, BARC-Satt309, distinguished most, if not all, SCN-susceptible genotypes from those carrying resistance at rhg1 derived from the important SCN-resistance sources ’Peking’, PI 437654, and PI 90763. BARC-Satt309 was also effective in distinguishing SCN resistance sources PI 88788 and PI 209332 from many, but not all, susceptible genotypes. BARC-Satt309 cannot be used in marker-assisted selection in populations developed from typical southern US cultivars crossed with the important resistance sources PI 88788 or PI 209332 because these genotypes all carry the identical allele at the BARC-Satt309 locus. A second SSR locus, BARC-Sat_168, was developed from a bacterial artificial chromosome (BAC) clone that was identified using the primers to BARC-Satt309. BARC-Sat_168 distinguished PI 88788 and PI 209332 from southern US cultivars such as ’Lee’, ’Bragg’ and ’Essex’. Both BARC-Satt309 and BARC-Sat_168 were used to assay lines from SCN-susceptible×SCN-resistant crosses and proved to be highly effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN susceptibility at the rhg1 locus. Received: 5 November 1998 / Accepted: 3 February 1999     

A recessive soybean cyst nematode allele for incompatibility with soybean PI 88287

The objective of this research was to characterise the degree of dominance of a soybean cyst nematode (Heterodera glycines) allele for incompatibility which interacts with a recessive soybean (Glycine max) allele for incompatibility to prevent the formation of cysts. Crosses of inbred nematode populations were made and the F, and F, populations evaluated for the numbers of cysts they could produce on several soybean lines. The nematode gene for avirulence interacts with the one recessive gene for resistance in soybean line PI 88287 and also appears to be recessive. This is the first example of a recessive-recessive gene-for-gene interaction; genes for avirulence and resistance are usually dominant. The difficulties of doing definitive genetic studies with cyst nematodes are discussed.