Active and inactive forms of biotin synthase occur in Heterodera glycines

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Active and inactive forms of biotin synthase occur in Heterodera glycines

Khee Man Kwon * / Sadia Bekal / Leslie L. Domier / Kris N. Lambert

Keywords : Biochemistry, Biotin, Biotin synthase, Dethiobiotin, Heterodera glycines , Horizontal gene transfer, Soybean cyst nematode, Vitamin B7

Citation Information : Journal of Nematology. Volume 51, Pages 1-12, DOI: https://doi.org/10.21307/jofnem-2019-069

License : (CC-BY-4.0)

Received Date : 29-April-2019 / Published Online: 25-October-2019

ARTICLE

ABSTRACT

Heterodera glycines, the soybean cyst nematode (SCN), is a plant-parasitic nematode capable of manipulating host plant biochemistry and development. Many studies have suggested that the nematode has acquired genes from bacteria via horizontal gene transfer events (HGTs) that have the potential to enhance nematode parasitism. A recent allelic imbalance analysis identified two candidate virulence genes, which also appear to have entered the SCN genome through HGTs. One of the candidate genes, H. glycines biotin synthase (HgBioB), contained sequence polymorphisms between avirulent and virulent inbred SCN strains. To test the function of these HgBioB alleles, a complementation experiment using biotin synthase-deficient Escherichia coli was conducted. Here, we report that avirulent nematodes produce an active biotin synthase while virulent ones contain an inactive form of the enzyme. Moreover, sequencing analysis of HgBioB genes from SCN field populations indicates the presence of diverse mixture of HgBioB alleles with the virulent form being the most prevalent. We hypothesize that the mutations in the inactive HgBioB allele within the virulent SCN could result in a change in protein function that in some unknown way bolster its parasitic lifestyle.

Graphical ABSTRACT

The soybean cyst nematode (SCN), Heterodera glycines, is an obligate plant parasite that poses a serious threat to soybean production worldwide. It is the most damaging pest of soybean in the USA and is estimated to cause more yield loss than any other disease (Koenning and Wrather, 2010; Wrather et al., 2010; Allen et al., 2017). Yield losses are attributed to the fact that this nematode injects a complex mixture of proteins and metabolites into the plant root cells (Niblack et al., 2006) and transforms them into a metabolically active feeding structure called a syncytium (Hussey, 1989). The syncytium becomes a nutrient sink where the nematode feeds throughout its life cycle (Jones, 1981; Endo, 1998). This disrupts the plant’s vascular system and prevents normal functioning of the root system leading to lower soybean yields.

What makes matters worse is the nematode’s ability to persist in soils for 10 or more years even in the absence of a soybean host (Riggs, 2004). The most common and practical control strategy for SCN is to plant resistant cultivars and rotate to non-host crops (Niblack et al., 2006). Although the management of SCN has been initially successful thanks to the use of resistant cultivars, the repeated use of the same resistance source has selected for virulent nematode populations that can reproduce on resistant cultivars (Niblack and Riggs, 2004; Mitchum et al., 2007; Niblack et al., 2008; Mitchum, 2016; McCarville et al., 2017). Understanding how SCN evade or suppress host plant resistance at the molecular level may allow the development of more effective sources of SCN-resistant soybean.

To this end, tremendous effort was put in the past decades to understand the complex interactions between the nematode and its host plant. The majority of studies on plant-parasitic nematode-host interactions were mainly focused on discovering genes that code for secreted effector proteins originating from the esophageal glands involved in manipulating host plant biochemistry, development, and defenses (Hussey, 1989; Smant et al., 1998; Lambert et al., 1999; Davis et al., 2000). Not all nematode secretory proteins, however, originate from the esophageal glands and some of the genes that encode secretory proteins can act as either virulence or avirulence genes. An esophageal gland-secreted protein/enzyme chorismate mutase (CM) is thought to suppress host defense by lowering the levels of chorismate-derived compounds required for plant defense (Lambert et al., 1999; Doyle and Lambert, 2003). Some SCN CM genes contain polymorphisms correlating with virulence on resistant soybean cultivars (Bekal et al., 2003; Lambert et al., 2005), also implicating these genes in SCN virulence. Similarly, a cuticle-secreted lipid-binding protein Gp-FAR-1 from Globodera pallida may be able to inhibit the host’s jasmonic acid signaling pathway (Prior et al., 2001). In contrast, the SPRYSEC effector protein Gp-RBP-1 secreted from an esophageal gland in G. pallida (Sacco et al., 2009), the venom allergen-like effector protein Gr-VAP1 secreted from an esophageal gland in G. rostochiensis (Lozano-Torres et al., 2012), MAP-1 secreted from amphids in Meloidogyne incognita (Semblat et al., 2001), and a SNARE-like protein (HgSLP-1) secreted from an esophageal gland in H. glycines (Bekal et al., 2015) appear to act as avirulence genes. Although not a secreted protein, Cg-1 from M. javanica is also thought to function as an avirulence gene (Gleason et al., 2008).

Some of the secreted proteins are closely related to bacterial proteins, suggesting that they have been acquired from bacteria through horizontal gene transfer events (HGTs) (Smant et al., 1998; Lambert et al., 1999; Davis et al., 2000; Bird et al., 2003; Bekal et al., 2015). While these studies focusing on secreted proteins and HGT candidates have been productive for understanding the molecular basis of nematode-host interactions, non-secreted proteins, enzymes, and metabolites have been overlooked; hence, horizontally transferred genes coding for non-secreted proteins/enzymes with bacterial homology could also play a critical role in SCN biology, parasitism, and (a)virulence.

Studies on non-secreted plant-parasitic nematode proteins have been few in number until Craig et al. (2008, 2009) showed that a certain group of non-secreted HGT proteins can include enzymes involved in vitamin B1, B5, B6, and B7 biosynthesis and their corresponding salvage pathways. More recently, a whole genome allelic imbalance analysis, conducted for the purpose of identifying single nucleotide polymorphisms (SNPs) associated with SCN genes implicated in virulence, found two candidate virulence genes, HgSLP-1 and biotin synthase (HgBioB), both of which have been acquired through HGTs (Bekal et al., 2015). HgBioB is a non-secreted protein/enzyme responsible for catalyzing the conversion of dethiobiotin (desthiobiotin or DTB) to biotin (vitamin B7). Furthermore, HgBioB contained SNPs that generated amino acid polymorphisms between avirulent and virulent inbred SCN strains, suggesting the allelic forms of HgBioB may have different activity (Bekal et al., 2015).

We postulated that these sequence polymorphisms could alter HgBioB activity and play an important role in SCN parasitism or virulence. Because it seemed reasonable that a nematode’s ability to produce more biotin might counteract a plant resistance mechanism that restricts the nematode from accessing free biotin or its immediate precursor, we hypothesized that virulent nematodes, which can reproduce on resistant cultivars, might produce a more active form of HgBioB than their avirulent counterparts, which cannot parasitize the resistant hosts. To support or refute this hypothesis, we tested the function of the two HgBioB alleles to ascertain if any functional differences could be detected between the virulent and avirulent forms of the enzyme.

Materials and methods

Construction and cloning of recombinant plasmids containing HgBioB

Two different DNA fragments HgBioB-avr (avirulent; accession number MH251885) and HgBioB-vir (virulent; accession number MH251886) that code for HgBioB in inbred SCN strains TN10 (avirulent; Hg-type 0; grown on Roma tomato) and TN20 (virulent; Hg-type 1, 2, 3, 4, 5, 6, 7; grown on plant introduction 437654), respectively, were synthesized with codons optimized for E. coli expression (Life Technologies, Carlsbad, CA). Also, two different mutagenized forms of HgBioB-vir, HgBioB-vir-M1 (virulent, mutagenized allele 1; accession number MH251887) and HgBioB-vir-M2 (virulent, mutagenized allele 2; accession number MH251888), were prepared by generating (i) an alanine to proline mutation (M1) at amino acid position 24 (A24P); and (ii) a glutamine to arginine mutation (M2) at amino acid position 44 (Q44R), respectively. M1 was produced via site-directed mutagenesis following the manufacturer’s instructions for In-Fusion cloning (Clontech, Mountain View, CA), using HgBioB-vir as the template. A pair of inverse polymerase chain reaction (PCR) primers (Table 1), BioB-mut1-F and BioB-mut1-R, was designed to share a 15-bp homology with each other and include a single-base substitution to generate an A24P mutation. Unlike M1, M2 was prepared by custom-ordering the synthetic DNA fragment containing the desired Q44R mutation (Life Technologies).

Table 1

Oligonucleotides for plasmid construction and sequencing analysis.

10.21307_jofnem-2019-069-t001.jpg

To perform In-Fusion cloning (Clontech), two pairs of PCR primers (Table 1) were designed for the insert and the vector so that they would share a 15-bp homology with each other. All four DNA fragments were amplified using the PCR primers BioB-F and BioB-R; and pBAD.LIC.8A cloning vector (Addgene plasmid No. 37501) was amplified with PCR primers BioB-Vec-F and BioB-Vec-R. All PCRs were done using an iProof HF Mastermix (Bio-Rad, Hercules, CA) following the manufacturer’s recommended PCR conditions. The resulting PCR products were gel-purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), joined together using an In-Fusion HD Cloning Kit (Clontech), and transformed into Stellar Competent Cells (Clontech) following the manufacturer’s instructions. Cells containing successful transformants were screened on Luria-Bertani (LB) agar plates with ampicillin (100 mg/ml) and a single colony was picked to inoculate 5 ml of LB broth with ampicillin (100 mg/ml) which was incubated overnight at 37°C. Recombinant plasmid DNA was extracted from the overnight culture using a QIAprep Spin Miniprep Kit (Qiagen) and the resulting plasmids were sequenced for verification at the University of Illinois Roy J. Carver Biotechnology Center.

Complementation of ΔbioB Escherichia coli with avirulent and virulent alleles of HgBioB

A kanamycin-resistant E. coli vitamin B7 auxotroph (K-12 Keio Collection: JW0758-1) was obtained from the Coli Genetic Stock Center at Yale University and used to prepare electrocompetent cells as described by Dower et al. (1988) and Hanahan (1983). Four different recombinant plasmids – pBAD.LIC.8A:: HgBioB-avr, pBAD.LIC.8A:: HgBioB-vir, pBAD.LIC.8A:: HgBioB-vir-M1, and pBAD.LIC.8A:: HgBioB-vir-M2 – were generated and independently transformed into the kanamycin-resistant ΔbioB mutant electrocompetent cells. For control purposes, an empty vector (pBAD.LIC.8 A) lacking any inserts was also transformed into the electrocompetent cells. The resulting transformants were screened on LB agar plates with ampicillin (100 mg/ml) and kanamycin (50 mg/ml), and a single colony was picked to inoculate 10 ml of LB broth with ampicillin (100 mg/ml) and kanamycin (50 mg/ml) for overnight incubation at 37°C. The overnight cultures of these four strains were thoroughly washed in liquid M9 minimal media to remove trace amounts of biotin that may be present, and then streaked onto M9 minimal media plates containing ampicillin (100 mg/ml), kanamycin (50 mg/ml), and either avidin or biotin. Plates containing avidin (1 mg/ml) were prepared to sequester trace amounts of biotin in the M9 media. Positive control plates were M9 media supplemented with biotin to a final concentration of 0.2 mM, and a negative control plate consisted of ΔbioB E. coli transformed with an empty pBAD.LIC.8A vector plated on M9 media lacking biotin. Some plates were also treated with 2% arabinose to induce HgBioB expression and monitor its potential effect on E. coli growth. All plates were initially incubated at 28°C for 48 hr, and then allowed to grow at room temperature for 10 d. E. coli growth was monitored daily.

Nematode culture and DNA extraction of SCN field populations

A collection of soil samples containing diverse SCN field populations representative of the H. glycines diversity was randomly chosen and prepared for nematode culture. The majority of these soil samples was from Iowa, a kind gift from Dr Gregory Tylka at Iowa State University (Ames, IA), and some of them were from Illinois. At least two to three different field populations originating from the same geographical region (e.g., at least two to three field populations from the east central region) were randomly selected for cyst extraction. Cysts from each field population were collected as described by Niblack et al. (1993) and crushed over a 0.25-mm (60 mesh) sieve to release eggs. Eggs were suspended in 2% carboxymethyl cellulose solution for an easy delivery of an approximately equal number of eggs during inoculation. This egg suspension was used to inoculate soybean cv. Essex plants, previously pre-germinated and planted into 50-ml Falcon tubes with the absorbent wick described in Bekal et al. (2015). Inoculated plants were grown for 8 wk to allow eggs to hatch and infect the plants. The resulting cysts from each field population, approximately 2 to 200 cysts from each plant, were harvested in a 2-ml cryotube vial, and stored at −80°C until use. The cyst-containing vials were placed in a pre-cooled rack in liquid nitrogen and two stainless steel beads (3.2 mm diameter) were added into each vial. These frozen cysts were then freeze-fractured with a bead beater homogenizer and total nucleic acid was extracted using an RNeasy Mini Kit (Qiagen).

Sequencing and statistical analysis of different SCN populations

In addition to 30 DNA samples from field populations, 4 DNA samples from inbred SCN strains – 1 avirulent (OP25) and 3 virulent inbred strains (OP20, OP50, and TN20) – were also included in the analysis. The OP strains were previously maintained as described in Dong and Opperman (1997) by Dr Terry Niblack (Ohio State University, Columbus, OH), and TN20 was maintained on PI 437654. From each extracted 50-μl DNA sample from a single population, 2.5 μl was used as a template for amplification using a GenomiPhi Kit (GE Healthcare, Piscataway, NJ) following the manufacturer’s protocols. The amplified DNA was treated with ExoSAP-IT (Affymetrix, Santa Clara, CA) for the degradation of primers and nucleotides that may interfere with downstream applications. A set of PCR primers (Table 1), BioB-flank-F1a and BioB-flank-R1a, flanking the polymorphic region of the HgBioB gene was designed for PCR amplification and subsequent sequencing analysis. All PCRs were done using a CloneAmp Hi-Fi PCR Premix (Clontech) following the manufacturer’s recommended conditions. The resulting PCR product was gel-purified using a QIAquick Gel Extraction Kit (Qiagen) and then sent for sequencing at the University of Illinois Roy J. Carver Biotechnology Center. The sequences were aligned to the original HgBioB sequence using Sequencher (Gene Codes Corporation, Ann Arbor, MI), and the relative chromatogram peak heights corresponding to amino acid positions 24 and 44 were measured with a 15-cm ruler on the computer screen to estimate the percentage of each polymorphic nucleotide(s).

Peak heights were statistically analyzed using both descriptive and inferential methods. First, a box plot was generated to compare the median values and understand the distributional characteristics of these alleles at both polymorphic regions, and then a Wilcoxon signed rank test was performed using the SAS Proc Univariate procedure (SAS Institute, Cary, NC) to test if the differences between avirulent and virulent alleles were statistically significant. For the signed rank test, a new variable ‘diff’ was set as the difference between the virulent and avirulent alleles (diff = vir − avir) at each amino acid for each SCN sample prior to running the Proc Univariate procedure on that variable. Finally, a Spearman rank-order correlation was conducted using the SAS Proc Corr procedure (SAS Institute) to calculate the coefficients for the relationship between the two variables, amino acid positions 24 and 44, for each set of virulent and avirulent pairs.

Results

Complementation of ΔbioB E. coli with avirulent and virulent alleles of HgBioB

We had observed differences in the predicted amino acid sequences between avirulent and virulent HgBioB alleles (Bekal et al., 2015), and thus hypothesized that such differences could lead to an altered enzyme function. To test for HgBioB function, we opted to use genetic complementation of a BioB-deficient E. coli mutant. Specifically, we wanted to learn if amino acid(s) changes, P24A or R44Q, the only two amino acids that are different between the HgBioB alleles, would alter BioB enzyme function.

To conduct this experiment, four plasmid constructs were generated to express the different forms of HgBioB; the virulent allele (HgBioB-vir, A24/Q44), the avirulent allele (HgBioB-avr, P24/R44), and the two single mutant alleles (HgBioB-vir-M1, P24/Q44; and HgBioB-vir-M2, A24/R44) (Fig. 1A). These two single mutants prepared from the virulent allele were included in the complementation studies to identify which amino acid location(s) were responsible for a functional enzyme activity. These plasmids, along with an empty vector control, were transformed into mutant E. coli lacking endogenous BioB function and were then allowed to grow on minimal agar with and without supplemented biotin.

Within 48 hr after streaking the E. coli containing the BioB constructs onto minimal media plates, all five biotin-supplemented plates (minimal media +biotin; positive controls) showed vigorous growth of E. coli as expected. Among the five plates lacking biotin (minimal media alone), however, growth only occurred for the bacteria containing the avirulent allele containing the P24/R44 amino acid combination (HgBioB-avr) (Fig. 1B). The other four constructs, E. coli carrying the empty vector (negative control), the virulent allele (HgBioB-vir), and the two mutagenized forms of the virulent allele (HgBioB-vir-M1 and HgBioB-vir-M2) failed to complement the growth of the mutant E. coli (Fig. 1B).

Our complementation test showed that avirulent SCN produces an active biotin synthase while virulent SCN contains an inactive form of the enzyme. Additionally, site-directed mutagenesis experiments confirmed that changing either of these two amino acids caused the HgBioB protein to become inactive, losing its ability to complement the mutant; therefore, a proline at position 24 (P24) and an arginine at position 44 (R44) are required for a functional BioB activity (Fig. 1A,B). This experiment was repeated and produced the same results.

Figure 1

Complementation by expression of HgBioB in biotin synthase-deficient E. coli strain. (A) HgBioB construct diagrams showing polymorphic amino acids, from N-terminus to C-terminus, at positions 24 and 44. HgBioB-vir was mutagenized to produce HgBioB-vir-M1, by generating an alanine to proline mutation at position 24 (A24P); and HgBioB-vir-M2, by generating a glutamine to arginine mutation at position 44 (Q44R). (B) The expression of the vector control, HgBioB-avr, HgBioB-vir, and two mutagenized forms of the virulent allele HgBioB-vir-M1 and HgBioB-vir-M2 on minimal media with and without supplemental biotin.

10.21307_jofnem-2019-069-f001.jpg

Sequencing, statistical analysis, and confirmation of SNPs from SCN field populations

Since our previous studies were based on just two inbred SCN strains, we were interested to learn if sequence variation in the HgBioB gene was common in different field populations of SCN. To assess how common HgBioB-vir alleles are in different populations of SCN, the HgBioB gene was PCR-amplified and sequenced from a series of SCN populations. Sequencing analysis of four inbred SCN strains (OP25, OP20, OP50, and TN20) showed that these strains were more likely to be uniform (i.e., have 100% allelic frequencies) carrying single homozygous forms (Table 2). In contrast, sequencing results from 30 SCN field populations showed that in most SCN populations, the HgBioB gene was not present in a single homozygous form, but rather in a diverse mixture consisting of both SNPs resulting in both active and inactive forms of HgBioB (Table 2). Statistical analysis of the field sequencing data was conducted by generating a box plot and comparing the median values. This analysis showed that the HgBioB-vir alleles were present in a higher percentage than the HgBioB-avr alleles at both polymorphic regions (i.e., more A24 than P24; more Q44 than R44) (Fig. 2A,B, Table 3). This trend was also supported by conducting a Wilcoxon signed rank test, the nonparametric alternative to the paired t-test, which was chosen because the polymorphic nucleotide percentage data for avirulent and virulent alleles were not normally distributed. The data were considered paired since we compared the two amino acid forms at each polymorphic region (i.e., A24 vs P24 and Q44 vs R44). The Wilcoxon signed rank test results showed that HgBioB-vir allele ranks were significantly higher than HgBioB-avr allele ranks for both amino acid positions 24 (Z=105, p<0.05) and 44 (Z=121.5, p<0.01). Therefore, from both descriptive and inferential statistical results, we concluded that the virulent alleles are more prevalent than the avirulent alleles in the field sites tested.

Table 2

Sequencing analysis of HgBioB nematode populations showing the percentage of amino acids at each polymorphic region.

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Table 3

Statistical analysis of amino acid percentages from nematode field populations.

10.21307_jofnem-2019-069-t003.jpg
Figure 2

Box plots of HgBioB amino acid polymorphism trends among SCN field populations, showing the percentage of amino acid differences for A24P (A) and Q44R (B).

10.21307_jofnem-2019-069-f002.jpg

In addition, it is interesting to note that these SNP changes occur simultaneously in pairs, at about the same frequency, between amino acid positions 24 and 44. For example, if a SNP in HgBioB results in a P24 then it would be paired with an R44; if it were an A24 then it would be paired with a Q44. This linkage of SNP frequency was supported by testing the correlations between the virulent allele pair (A24 and Q44) as well as the avirulent allele pair (P24 and R44) using a Spearman rank-order correlation, the nonparametric alternative to the Pearson product-moment correlation, chosen because the assumptions of normality could not be met. The Spearman correlation results showed that there was a positive correlation for both virulent (r=0.8683, n=30, p<0.001) and avirulent (r=0.9184, n=30, p<0.001) pairs. Scatterplots for both avirulent and virulent pairs also summarize that there is a positive linear relationship between amino acid positions 24 and 44 (Fig. 3A,B). The strong, positive correlation between these positions suggests that these amino acid changes must both occur in the same protein to exert their desired, but unknown function. Although a rarity, alternative SNPs that result in a threonine at position 24 (T24) and a proline at position 44 (P44) were detected as well, suggesting an important but unknown function of these two amino acid positions in the HgBioB enzyme.

Figure 3

Scatterplots showing the HgBioB avirulent and virulent polymorphic amino acid pairs. (A) The avirulent pair shows a positive linear relationship between amino acid positions P24 and R44. (B) Similarly, the virulent pair also shows a positive linear relationship between amino acid positions A24 and Q44.

10.21307_jofnem-2019-069-f003.jpg

Discussion

In this study, we compared the enzymatic activities of biotin synthase genes from virulent and avirulent inbred SCN strains and showed that the HgBioB-vir from virulent nematodes lacked activity while the HgBioB-avr from avirulent nematodes was fully active, suggesting HgBioB is important in SCN virulence. Biotin (vitamin B7 or vitamin H) is a member of the water-soluble vitamin B family and an essential enzyme cofactor, necessary for all three domains of life (Depeint et al., 2006; Zempleni et al., 2008; Waldrop et al., 2012). This vitamin acts as a carboxyl carrier for biotin-dependent carboxylases, decarboxylases, and transcarboxylases, which are critical for fatty acid, amino acid, and carbohydrate metabolic processes (Knowles, 1989; Alban et al., 2000; Attwood and Wallace, 2002; Cronan, 2014). Biotin is also involved in cell signaling, chromatin structure, and epigenetic gene regulation (Zempleni, 2005). More recent discovery shows that biotin plays a role in bacterial virulence (Feng et al., 2014), supporting its potential importance in nematode virulence and parasitism.

Despite its crucial functions, the ability to synthesize biotin de novo, either completely or partially, is only found in bacteria, plants, and some fungi (Hall and Dietrich, 2007; Alban, 2011). Most of these organisms synthesize biotin from pimeloyl-CoA (ACP) in a highly conserved four-step pathway that consists of 7-keto-8-aminopelargonic acid, 7,8-diaminopelargnoic acid, DTB, and biotin (Streit and Entcheva, 2003; Alban, 2011; Lin and Cronan, 2011). However, animals do not have the ability to synthesize biotin (Hall and Dietrich, 2007) and must acquire vitamins from their diet (Craig et al., 2008; Craig et al., 2009). Nevertheless, it is strange that SCN, an animal and a parasite, lost the ability to synthesize biotin and then re-acquired the biotin synthase gene via HGT, because the nematode should be able to acquire biotin from its host. It should be noted that SCN does not have the complete biotin biosynthesis pathway, but a partial pathway that encodes the last key enzyme, biotin synthase (Craig et al., 2009), which converts DTB to biotin (Cleary and Campbell, 1972). The presence of this incomplete de novo biotin biosynthesis pathway implies that biotin has an important function in SCN and suggests the nematode is scavenging DTB to make biotin from its host (Craig et al., 2009). Although free biotin can be present at high levels in plant cells, unlike animal cells which have more protein-bound biotin (Alban, 2011), it remains unknown if sufficient free biotin is available to SCN as they feed from syncytial cells of high metabolic activity. It is possible that SCN, a filter feeder that does not consume plants via chewing, cannot obtain enough biotin if it is bound to proteins. That is, their presumed prevalence for withdrawing nutrients via feeding tubes, which act as a molecular sieve (Böckenhoff and Grundler, 1994), may limit their vitamin ingestion; thus, having the biotin synthase gene, or other vitamin B biosynthetic genes, could be a selective advantage.

We previously predicted that virulent nematodes might gain a competitive advantage by producing a more enzymatically active biotin synthase (Bekal et al., 2015) and utilizing the precursors to expand its salvage pathways (Craig et al., 2008, 2009). In our complementation study, however, only the avirulent allele of HgBioB (HgBioB-avr) successfully complemented the growth of an E. coli strain containing a deletion of bioB. This showed that avirulent inbred SCN produces an active biotin synthase while virulent inbred nematodes contain an inactive form of the enzyme, thereby refuting our hypothesis.

However, it was possible that the inbred nature of the nematodes selected for a rare HgBioB allele that was inactive. To assess how common HgBioB-vir alleles are in natural populations of SCN, the HgBioB gene was PCR-amplified and sequenced from a series of nematode populations. The sequencing data showed that both SNPs were present in the field SCN populations, resulting in a mixture of both active and inactive forms of HgBioB, which is expected due to the heterozygous nature of most nematode populations. In most SCN populations, the virulent forms were prevalent, mirroring the increased prevalence of virulent SCN described by many nematologists (Niblack and Riggs, 2004; Mitchum et al., 2007; Niblack et al., 2008; Tylka and Mullaney, 2015). This implies that an opposing selection for HgBioB activity is occurring in the wild. It would be interesting to find a correlation, if any, between the prevalence of active/inactive forms of HgBioB with certain Hg-types in the field populations; however, we were not able to harvest enough cysts to carry out Hg-type bioassays and, therefore, all cysts that were harvested were used for the sequencing analysis. There also seems to be an important, but unknown reason why the SNP changes occur concurrently in pairs with similar frequencies between amino acid positions 24 and 44. Since the avirulent and virulent forms of the HgBioB alleles only differ at these two positions, we initially speculated that these amino acid sequence polymorphisms might affect the overall biotin synthase activity, especially if these SNPs were located close to the conserved regions responsible for substrate-binding or catalytic activity of the enzyme. However, these SNPs were not in the conserved C53xxxC57xxC60 motif (C, cysteine; x, any amino acid), a key characteristic of biotin synthase and other enzymes in the radical S-adenosyl-methionine superfamily (Frey and Booker, 2001; Jarrett, 2003; Berkovitch et al., 2004). Both the presence of sequence polymorphisms at amino acid positions 24 and 44, and the evidence from the site-directed mutagenesis indicating the absolute requirement for both proline and arginine at those specific locations (P24 and R44) further stress the importance of these amino acids in spite of their unknown functional role in the HgBioB enzyme. Since our sequencing data showed that these SNPs did not occur randomly but changed in a coordinated fashion, these SNPs might lead to a complete change in protein function other than biotin synthase. Because the nematode encodes the last key enzyme involved in the biotin biosynthesis pathway, the nematode could have repurposed the biotin synthase enzyme for a completely different task. It has been reported that the housekeeping glutathione synthetase (GS) gene from G. pallida was repurposed to diversify its biochemical function, forming a novel class of GS-like enzymes (Lilley et al., 2018). In a similar manner, it is possible that the biotin synthase gene may also modify its biochemical activity to produce a new family of biotin synthase-like enzymes for the evolution of its parasitic lifestyle.

It is also possible that these SNPs might convert the HgBioB enzyme into a biotin-related toxin-binding protein that may protect the virulent nematode from host defense mechanisms. Such biotin-related anti-metabolites do occur in bacteria (Hanka et al., 1966, 1969, 1972), but it is unclear if such toxins play a role in SCN resistance. Regardless of the role the inactive form of HgBioB plays in the SCN life cycle, it would have to provide a survival advantage to the nematode to counteract any negative effects such as slower growth or lower reproduction due to the absence of biotin scavenging ability of an active HgBioB. It has been shown that a reproductive fitness cost is associated with virulence in M. incognita (Castagnone-Sereno et al., 2007; Djian-Caporalino et al., 2011), and therefore this could also be applicable to virulent SCN where low biotin availability could cause biotin deficiency, which may result in low reproductive fitness.

In the future, it would be interesting to see if virulent SCN also have a reproductive fitness cost by correlating a nematode’s biotin synthase activity or its biotin content with its reproductive index and/or cyst size. Further study of the biotin synthase genes from other SCN strains and even from other plant-parasitic nematodes may help elucidate how the loss of HgBioB activity might benefit SCN parasitism and/or virulence, which may lead to the development of novel methods to minimize soybean yield losses affected by these devastating pathogens.

Acknowledgements

The authors thank Dr Gregory Tylka (Iowa State University, Ames, IA) for providing the SCN field samples from Iowa. The authors also thank Dr Alex Lipka (University of Illinois, Urbana, IL) for his professional advice on statistical analysis. This study was supported by the United Soybean Board (Proposal Number: 1620-532-5620). The authors have no conflict of interest to declare.

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  3. Allen, T. W. , Bradley, C. A. , Sisson, A. J. , Byamukama, E. , Chilvers, M. I. , Coker, C. M. , Collins, A. A. , Damicone, J. P. , Dorrance, A. E. and Dufault, N. S. 2017. Soybean yield loss estimates due to diseases in the United States and Ontario, Canada, from 2010 to 2014. Plant Health Progress 18: 19–27.
  4. Attwood, P. V. and Wallace, J. C. 2002. Chemical and catalytic mechanisms of carboxyl transfer reactions in biotin-dependent enzymes. Accounts of Chemical Research 35: 113–120.
  5. Bekal, S. , Niblack, T. L. and Lambert, K. N. 2003. A chorismate mutase from the soybean cyst nematode Heterodera glycines shows polymorphisms that correlate with virulence. Molecular Plant-Microbe Interactions 16: 439–446.
  6. Bekal, S. , Domier, L. L. , Gonfa, B. , Lakhssassi, N. , Meksem, K. and Lambert, K. N. 2015. A SNARE-like protein and biotin are implicated in soybean cyst nematode virulence. PLoS One 10: e0145601.
  7. Berkovitch, F. , Nicolet, Y. , Wan, J. T. , Jarrett, J. T. and Drennan, C. L. 2004. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303: 76–79.
  8. Bird, D. M. , Opperman, C. H. and Davies, K. G. 2003. Interactions between bacteria and plant-parasitic nematodes: now and then. International Journal for Parasitology 33: 1269–1276.
  9. Böckenhoff, A. and Grundler, F. 1994. Studies on the nutrient uptake by the beet cyst nematode Heterodera schachtii by in situ microinjection of fluorescent probes into the feeding structures in Arabidopsis thaliana . Parasitology 109: 249–255.
  10. Castagnone-Sereno, P. , Bongiovanni, M. and Wajnberg, E. 2007. Selection and parasite evolution: a reproductive fitness cost associated with virulence in the parthenogenetic nematode Meloidogyne incognita . Evolutionary Ecology 21: 259–270.
  11. Cleary, P. P. and Campbell, A. 1972. Deletion and complementation analysis of the biotin gene cluster of Escherichia coli . Journal of Bacteriology 112: 830–839.
  12. Craig, J. P. , Bekal, S. , Niblack, T. , Domier, L. and Lambert, K. N. 2009. Evidence for horizontally transferred genes involved in the biosynthesis of vitamin b(1), b(5), and b(7) in Heterodera glycines . Journal of Nematology 41: 281–290.
  13. Craig, J. P. , Bekal, S. , Hudson, M. , Domier, L. , Niblack, T. and Lambert, K. N. 2008. Analysis of a horizontally transferred pathway involved in vitamin b6 biosynthesis from the soybean cyst nematode Heterodera glycines . Molecular Biology and Evolution 25: 2085–2098.
  14. Cronan, J. E. 2014. Biotin and lipoic acid: synthesis, attachment and regulation. EcoSal Plus 6(1) :6.
  15. Davis, E. L. , Hussey, R. S. , Baum, T. J. , Bakker, J. , Schots, A. , Rosso, M.- N. and Abad, P. 2000. Nematode parasitism genes. Annual Review of Phytopathology 38: 365–396.
  16. Depeint, F. , Bruce, W. R. , Shangari, N. , Mehta, R. and O’brien, P. J. 2006. Mitochondrial function and toxicity: role of b vitamins on the one-carbon transfer pathways. Chemico-Biological Interactions 163: 113–132.
  17. Djian-Caporalino, C. , Molinari, S. , Palloix, A. , Ciancio, A. , Fazari, A. , Marteu, N. , Ris, N. and Castagnone-Sereno, P. 2011. The reproductive potential of the root-knot nematode Meloidogyne incognita is affected by selection for virulence against major resistance genes from tomato and pepper. European Journal of Plant Pathology 131: 431.
  18. Dong, K. and Opperman, C. H. 1997. Genetic analysis of parasitism in the soybean cyst nematode Heterodera glycines . Genetics 146: 1311–1318.
  19. Dower, W. J. , Miller, J. F. and Ragsdale, C. W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Research 16: 6127–6145.
  20. Doyle, E. A. and Lambert, K. N. 2003. Meloidogyne javanica chorismate mutase 1 alters plant cell development. Molecular Plant-Microbe Interactions 16: 123–131.
  21. Endo, B. Y. 1998. Atlas on ultrastructure of infective juveniles of the soybean cyst nematode, Heterodera glycines. United States Department of Agriculture, Washington, DC, 215–220.
  22. Feng, Y. , Napier, B. A. , Manandhar, M. , Henke, S. K. , Weiss, D. S. and Cronan, J. E. 2014. A Francisella virulence factor catalyses an essential reaction of biotin synthesis. Molecular Microbiology 91: 300–314.
  23. Frey, P. A. and Booker, S. J. 2001. Radical mechanisms of S-adenosylmethionine-dependent enzymes. Advances in Protein Chemistry 58: 1–45.
  24. Gleason, C. A. , Liu, Q. L. and Williamson, V. M. 2008. Silencing a candidate nematode effector gene corresponding to the tomato resistance gene Mi-1 leads to acquisition of virulence. Molecular Plant-Microbe Interactions 21: 576–585.
  25. Hall, C. and Dietrich, F. S. 2007. The reacquisition of biotin prototrophy in Saccharomyces cerevisiae involved horizontal gene transfer, gene duplication and gene clustering. Genetics 177: 2293–2307.
  26. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166: 557–580.
  27. Hanka, L. , Bergey, M. and Kelly, R. 1966. Naturally occurring antimetabolite antibiotic related to biotin. Science 154: 1667–1668.
  28. Hanka, L. , Martin, D. and Reineke, L. 1972. Two new antimetabolites of biotin: α-methyldethiobiotin and α-methylbiotin. Antimicrobial Agents and Chemotherapy 1: 135–138.
  29. Hanka, L. , Reineke, L. and Martin, D. 1969. Biological studies with α-dehydrobiotin. Journal of Bacteriology 100: 42–46.
  30. Hussey, R. S. 1989. Disease-inducing secretions of plant-parasitic nematodes. Annual Review of Phytopathology 27: 123–141.
  31. Jarrett, J. T. 2003. The generation of 5’-deoxyadenosyl radicals by adenosylmethionine-dependent radical enzymes. Current Opinion in Chemical Biology 7: 174–182.
  32. Jones, M. 1981. Host cell responses to endoparasitic nematode attack: structure and function of giant cells and syncytia. Annals of Applied Biology 97: 353–372.
  33. Knowles, J. R. 1989. The mechanism of biotin-dependent enzymes. Annual Review of Biochemistry 58: 195–221.
  34. Koenning, S. R. and Wrather, J. A. 2010. Suppression of soybean yield potential in the continental United States by plant diseases from 2006 to 2009. Plant Health Progress, p. 10.
  35. Lambert, K. N. , Allen, K. D. and Sussex, I. M. 1999. Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica . Molecular Plant-Microbe Interactions 12: 328–336.
  36. Lambert, K. N. , Bekal, S. , Domier, L. L. , Niblack, T. L. , Noel, G. R. and Smyth, C. A. 2005. Selection of Heterodera glycines chorismate mutase-1 alleles on nematode-resistant soybean. Molecular Plant-Microbe Interactions 18: 593–601.
  37. Lilley, C. J. , Maqbool, A. , Wu, D. , Yusup, H. B. , Jones, L. M. , Birch, P. R. , Banfield, M. J. , Urwin, P. E. and Eves-Van Den Akker, S. 2018. Effector gene birth in plant parasitic nematodes: neofunctionalization of a housekeeping glutathione synthetase gene. PLoS Genetics 14: e1007310.
  38. Lin, S. and Cronan, J. E. 2011. Closing in on complete pathways of biotin biosynthesis. Molecular BioSystems 7: 1811–1821.
  39. Lozano-Torres, J. L. , Wilbers, R. H. , Gawronski, P. , Boshoven, J. C. , Finkers-Tomczak, A. , Cordewener, J. H. , America, A. H. , Overmars, H. A. , Van’T Klooster, J. W. and Baranowski, L. 2012. Dual disease resistance mediated by the immune receptor cf-2 in tomato requires a common virulence target of a fungus and a nematode. Proceedings of the National Academy of Sciences 109: 10119–10124.
  40. McCarville, M. T. , Marett, C. C. , Mullaney, M. P. , Gebhart, G. D. and Tylka, G. L. 2017. Increase in soybean cyst nematode virulence and reproduction on resistant soybean varieties in Iowa from 2001 to 2015 and the effects on soybean yields. Plant Health Progress 18: 146–155.
  41. Mitchum, M. G. 2016. Soybean resistance to the soybean cyst nematode Heterodera glycines: an update. Phytopathology 106: 1444–1450.
  42. Mitchum, M. G. , Wrather, J. A. , Heinz, R. D. , Shannon, J. G. and Danekas, G. 2007. Variability in distribution and virulence phenotypes of Heterodera glycines in Missouri during 2005. Plant Disease 91: 1473–1476.
  43. Niblack, T. and Riggs, R. D. 2004. Variation in virulence phenotypes. in Schmitt, D. P. , Wrather, J. A. and Riggs, R. D. . (Eds), Biology and management of soybean cyst nematode 2nd ed. Schmitt & Associates of Marceline, Marceline, MO, 57–71.
  44. Niblack, T. , Lambert, K. and Tylka, G. 2006. A model plant pathogen from the Kingdom Animalia: Heterodera glycines, the soybean cyst nematode. Annual Review of Phytopathology 44: 283–303.
  45. Niblack, T. , Colgrove, A. , Colgrove, K. and Bond, J. 2008. Shift in virulence of soybean cyst nematode is associated with use of resistance from PI 88788. Plant Health Progress 0118–0101.
  46. Niblack, T. , Heinz, R. , Smith, G. and Donald, P. 1993. Distribution, density, and diversity of Heterodera glycines in Missouri. Journal of Nematology 25: 880.
  47. Prior, A. , Jones, J. T. , Blok, V. C. , Beauchamp, J. , Mcdermott, L. , Cooper, A. and Kennedy, M. W. 2001. A surface-associated retinol-and fatty acid-binding protein (Gp-far-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern. Biochemical Journal 356: 387.
  48. Riggs, R. D. 2004. History and distribution. in Schmitt, D. P. , Wrather, J. A. and Riggs, R. D. . (Eds), Biology and management of soybean cyst nematode 2nd ed. Schmitt & Associates of Marceline, Marceline, MO, 9–40.
  49. Sacco, M. A. , Koropacka, K. , Grenier, E. , Jaubert, M. J. , Blanchard, A. , Goverse, A. , Smant, G. and Moffett, P. 2009. The cyst nematode SPRYSEC protein rbp-1 elicits gpa2-and rangap2-dependent plant cell death. PLoS Pathogens 5: e1000564.
  50. Semblat, J.- P. , Rosso, M.- N. , Hussey, R. S. , Abad, P. and Castagnone-Sereno, P. 2001. Molecular cloning of a cDNA encoding an amphid-secreted putative avirulence protein from the root-knot nematode Meloidogyne incognita . Molecular Plant-Microbe Interactions 14: 72–79.
  51. Smant, G. , Stokkermans, J. P. , Yan, Y. , De Boer, J. M. , Baum, T. J. , Wang, X. , Hussey, R. S. , Gommers, F. J. , Henrissat, B. and Davis, E. L. 1998. Endogenous cellulases in animals: isolation of β-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes. Proceedings of the National Academy of Sciences 95: 4906–4911.
  52. Streit, W. and Entcheva, P. 2003. Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production. Applied Microbiology and Biotechnology 61: 21–31.
  53. Tylka, G. and Mullaney, M. 2015. Soybean cyst nematode-resistant soybeans for Iowa. Extension publication PM 1649, Iowa State University Extension, Ames, IA.
  54. Waldrop, G. L. , Holden, H. M. and Maurice, M. S. 2012. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms. Protein Science 21: 1597–1619.
  55. Wrather, A. , Shannon, G. , Balardin, R. , Carregal, L. , Escobar, R. , Gupta, G. , Ma, Z. , Morel, W. , Ploper, D. and Tenuta, A. 2010. Effect of diseases on soybean yield in the top eight producing countries in 2006. Plant Health Progress 10: 1094.
  56. Zempleni, J. 2005. Uptake, localization, and noncarboxylase roles of biotin. Annual Review of Nutrition 25: 175–196.
  57. Zempleni, J. , Hassan, Y. I. and Wijeratne, S. S. 2008. Biotin and biotinidase deficiency. Expert Review of Endocrinology & Metabolism 3: 715–724.
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FIGURES & TABLES

Figure 1

Complementation by expression of HgBioB in biotin synthase-deficient E. coli strain. (A) HgBioB construct diagrams showing polymorphic amino acids, from N-terminus to C-terminus, at positions 24 and 44. HgBioB-vir was mutagenized to produce HgBioB-vir-M1, by generating an alanine to proline mutation at position 24 (A24P); and HgBioB-vir-M2, by generating a glutamine to arginine mutation at position 44 (Q44R). (B) The expression of the vector control, HgBioB-avr, HgBioB-vir, and two mutagenized forms of the virulent allele HgBioB-vir-M1 and HgBioB-vir-M2 on minimal media with and without supplemental biotin.

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Figure 2

Box plots of HgBioB amino acid polymorphism trends among SCN field populations, showing the percentage of amino acid differences for A24P (A) and Q44R (B).

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Figure 3

Scatterplots showing the HgBioB avirulent and virulent polymorphic amino acid pairs. (A) The avirulent pair shows a positive linear relationship between amino acid positions P24 and R44. (B) Similarly, the virulent pair also shows a positive linear relationship between amino acid positions A24 and Q44.

Full Size   |   Slide (.pptx)

REFERENCES

  1. Alban, C. 2011. Biotin (vitamin B8) synthesis in plants. in Rébeillé, F. , Douce, R. , Kader, J. -C. and Delseny, M. . (Eds), Advances in botanical research 59 Academic Press, London,39–66.
  2. Alban, C. , Job, D. and Douce, R. 2000. Biotin metabolism in plants. Annual Review of Plant Biology 51: 17–47.
  3. Allen, T. W. , Bradley, C. A. , Sisson, A. J. , Byamukama, E. , Chilvers, M. I. , Coker, C. M. , Collins, A. A. , Damicone, J. P. , Dorrance, A. E. and Dufault, N. S. 2017. Soybean yield loss estimates due to diseases in the United States and Ontario, Canada, from 2010 to 2014. Plant Health Progress 18: 19–27.
  4. Attwood, P. V. and Wallace, J. C. 2002. Chemical and catalytic mechanisms of carboxyl transfer reactions in biotin-dependent enzymes. Accounts of Chemical Research 35: 113–120.
  5. Bekal, S. , Niblack, T. L. and Lambert, K. N. 2003. A chorismate mutase from the soybean cyst nematode Heterodera glycines shows polymorphisms that correlate with virulence. Molecular Plant-Microbe Interactions 16: 439–446.
  6. Bekal, S. , Domier, L. L. , Gonfa, B. , Lakhssassi, N. , Meksem, K. and Lambert, K. N. 2015. A SNARE-like protein and biotin are implicated in soybean cyst nematode virulence. PLoS One 10: e0145601.
  7. Berkovitch, F. , Nicolet, Y. , Wan, J. T. , Jarrett, J. T. and Drennan, C. L. 2004. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303: 76–79.
  8. Bird, D. M. , Opperman, C. H. and Davies, K. G. 2003. Interactions between bacteria and plant-parasitic nematodes: now and then. International Journal for Parasitology 33: 1269–1276.
  9. Böckenhoff, A. and Grundler, F. 1994. Studies on the nutrient uptake by the beet cyst nematode Heterodera schachtii by in situ microinjection of fluorescent probes into the feeding structures in Arabidopsis thaliana . Parasitology 109: 249–255.
  10. Castagnone-Sereno, P. , Bongiovanni, M. and Wajnberg, E. 2007. Selection and parasite evolution: a reproductive fitness cost associated with virulence in the parthenogenetic nematode Meloidogyne incognita . Evolutionary Ecology 21: 259–270.
  11. Cleary, P. P. and Campbell, A. 1972. Deletion and complementation analysis of the biotin gene cluster of Escherichia coli . Journal of Bacteriology 112: 830–839.
  12. Craig, J. P. , Bekal, S. , Niblack, T. , Domier, L. and Lambert, K. N. 2009. Evidence for horizontally transferred genes involved in the biosynthesis of vitamin b(1), b(5), and b(7) in Heterodera glycines . Journal of Nematology 41: 281–290.
  13. Craig, J. P. , Bekal, S. , Hudson, M. , Domier, L. , Niblack, T. and Lambert, K. N. 2008. Analysis of a horizontally transferred pathway involved in vitamin b6 biosynthesis from the soybean cyst nematode Heterodera glycines . Molecular Biology and Evolution 25: 2085–2098.
  14. Cronan, J. E. 2014. Biotin and lipoic acid: synthesis, attachment and regulation. EcoSal Plus 6(1) :6.
  15. Davis, E. L. , Hussey, R. S. , Baum, T. J. , Bakker, J. , Schots, A. , Rosso, M.- N. and Abad, P. 2000. Nematode parasitism genes. Annual Review of Phytopathology 38: 365–396.
  16. Depeint, F. , Bruce, W. R. , Shangari, N. , Mehta, R. and O’brien, P. J. 2006. Mitochondrial function and toxicity: role of b vitamins on the one-carbon transfer pathways. Chemico-Biological Interactions 163: 113–132.
  17. Djian-Caporalino, C. , Molinari, S. , Palloix, A. , Ciancio, A. , Fazari, A. , Marteu, N. , Ris, N. and Castagnone-Sereno, P. 2011. The reproductive potential of the root-knot nematode Meloidogyne incognita is affected by selection for virulence against major resistance genes from tomato and pepper. European Journal of Plant Pathology 131: 431.
  18. Dong, K. and Opperman, C. H. 1997. Genetic analysis of parasitism in the soybean cyst nematode Heterodera glycines . Genetics 146: 1311–1318.
  19. Dower, W. J. , Miller, J. F. and Ragsdale, C. W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Research 16: 6127–6145.
  20. Doyle, E. A. and Lambert, K. N. 2003. Meloidogyne javanica chorismate mutase 1 alters plant cell development. Molecular Plant-Microbe Interactions 16: 123–131.
  21. Endo, B. Y. 1998. Atlas on ultrastructure of infective juveniles of the soybean cyst nematode, Heterodera glycines. United States Department of Agriculture, Washington, DC, 215–220.
  22. Feng, Y. , Napier, B. A. , Manandhar, M. , Henke, S. K. , Weiss, D. S. and Cronan, J. E. 2014. A Francisella virulence factor catalyses an essential reaction of biotin synthesis. Molecular Microbiology 91: 300–314.
  23. Frey, P. A. and Booker, S. J. 2001. Radical mechanisms of S-adenosylmethionine-dependent enzymes. Advances in Protein Chemistry 58: 1–45.
  24. Gleason, C. A. , Liu, Q. L. and Williamson, V. M. 2008. Silencing a candidate nematode effector gene corresponding to the tomato resistance gene Mi-1 leads to acquisition of virulence. Molecular Plant-Microbe Interactions 21: 576–585.
  25. Hall, C. and Dietrich, F. S. 2007. The reacquisition of biotin prototrophy in Saccharomyces cerevisiae involved horizontal gene transfer, gene duplication and gene clustering. Genetics 177: 2293–2307.
  26. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166: 557–580.
  27. Hanka, L. , Bergey, M. and Kelly, R. 1966. Naturally occurring antimetabolite antibiotic related to biotin. Science 154: 1667–1668.
  28. Hanka, L. , Martin, D. and Reineke, L. 1972. Two new antimetabolites of biotin: α-methyldethiobiotin and α-methylbiotin. Antimicrobial Agents and Chemotherapy 1: 135–138.
  29. Hanka, L. , Reineke, L. and Martin, D. 1969. Biological studies with α-dehydrobiotin. Journal of Bacteriology 100: 42–46.
  30. Hussey, R. S. 1989. Disease-inducing secretions of plant-parasitic nematodes. Annual Review of Phytopathology 27: 123–141.
  31. Jarrett, J. T. 2003. The generation of 5’-deoxyadenosyl radicals by adenosylmethionine-dependent radical enzymes. Current Opinion in Chemical Biology 7: 174–182.
  32. Jones, M. 1981. Host cell responses to endoparasitic nematode attack: structure and function of giant cells and syncytia. Annals of Applied Biology 97: 353–372.
  33. Knowles, J. R. 1989. The mechanism of biotin-dependent enzymes. Annual Review of Biochemistry 58: 195–221.
  34. Koenning, S. R. and Wrather, J. A. 2010. Suppression of soybean yield potential in the continental United States by plant diseases from 2006 to 2009. Plant Health Progress, p. 10.
  35. Lambert, K. N. , Allen, K. D. and Sussex, I. M. 1999. Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica . Molecular Plant-Microbe Interactions 12: 328–336.
  36. Lambert, K. N. , Bekal, S. , Domier, L. L. , Niblack, T. L. , Noel, G. R. and Smyth, C. A. 2005. Selection of Heterodera glycines chorismate mutase-1 alleles on nematode-resistant soybean. Molecular Plant-Microbe Interactions 18: 593–601.
  37. Lilley, C. J. , Maqbool, A. , Wu, D. , Yusup, H. B. , Jones, L. M. , Birch, P. R. , Banfield, M. J. , Urwin, P. E. and Eves-Van Den Akker, S. 2018. Effector gene birth in plant parasitic nematodes: neofunctionalization of a housekeeping glutathione synthetase gene. PLoS Genetics 14: e1007310.
  38. Lin, S. and Cronan, J. E. 2011. Closing in on complete pathways of biotin biosynthesis. Molecular BioSystems 7: 1811–1821.
  39. Lozano-Torres, J. L. , Wilbers, R. H. , Gawronski, P. , Boshoven, J. C. , Finkers-Tomczak, A. , Cordewener, J. H. , America, A. H. , Overmars, H. A. , Van’T Klooster, J. W. and Baranowski, L. 2012. Dual disease resistance mediated by the immune receptor cf-2 in tomato requires a common virulence target of a fungus and a nematode. Proceedings of the National Academy of Sciences 109: 10119–10124.
  40. McCarville, M. T. , Marett, C. C. , Mullaney, M. P. , Gebhart, G. D. and Tylka, G. L. 2017. Increase in soybean cyst nematode virulence and reproduction on resistant soybean varieties in Iowa from 2001 to 2015 and the effects on soybean yields. Plant Health Progress 18: 146–155.
  41. Mitchum, M. G. 2016. Soybean resistance to the soybean cyst nematode Heterodera glycines: an update. Phytopathology 106: 1444–1450.
  42. Mitchum, M. G. , Wrather, J. A. , Heinz, R. D. , Shannon, J. G. and Danekas, G. 2007. Variability in distribution and virulence phenotypes of Heterodera glycines in Missouri during 2005. Plant Disease 91: 1473–1476.
  43. Niblack, T. and Riggs, R. D. 2004. Variation in virulence phenotypes. in Schmitt, D. P. , Wrather, J. A. and Riggs, R. D. . (Eds), Biology and management of soybean cyst nematode 2nd ed. Schmitt & Associates of Marceline, Marceline, MO, 57–71.
  44. Niblack, T. , Lambert, K. and Tylka, G. 2006. A model plant pathogen from the Kingdom Animalia: Heterodera glycines, the soybean cyst nematode. Annual Review of Phytopathology 44: 283–303.
  45. Niblack, T. , Colgrove, A. , Colgrove, K. and Bond, J. 2008. Shift in virulence of soybean cyst nematode is associated with use of resistance from PI 88788. Plant Health Progress 0118–0101.
  46. Niblack, T. , Heinz, R. , Smith, G. and Donald, P. 1993. Distribution, density, and diversity of Heterodera glycines in Missouri. Journal of Nematology 25: 880.
  47. Prior, A. , Jones, J. T. , Blok, V. C. , Beauchamp, J. , Mcdermott, L. , Cooper, A. and Kennedy, M. W. 2001. A surface-associated retinol-and fatty acid-binding protein (Gp-far-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern. Biochemical Journal 356: 387.
  48. Riggs, R. D. 2004. History and distribution. in Schmitt, D. P. , Wrather, J. A. and Riggs, R. D. . (Eds), Biology and management of soybean cyst nematode 2nd ed. Schmitt & Associates of Marceline, Marceline, MO, 9–40.
  49. Sacco, M. A. , Koropacka, K. , Grenier, E. , Jaubert, M. J. , Blanchard, A. , Goverse, A. , Smant, G. and Moffett, P. 2009. The cyst nematode SPRYSEC protein rbp-1 elicits gpa2-and rangap2-dependent plant cell death. PLoS Pathogens 5: e1000564.
  50. Semblat, J.- P. , Rosso, M.- N. , Hussey, R. S. , Abad, P. and Castagnone-Sereno, P. 2001. Molecular cloning of a cDNA encoding an amphid-secreted putative avirulence protein from the root-knot nematode Meloidogyne incognita . Molecular Plant-Microbe Interactions 14: 72–79.
  51. Smant, G. , Stokkermans, J. P. , Yan, Y. , De Boer, J. M. , Baum, T. J. , Wang, X. , Hussey, R. S. , Gommers, F. J. , Henrissat, B. and Davis, E. L. 1998. Endogenous cellulases in animals: isolation of β-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes. Proceedings of the National Academy of Sciences 95: 4906–4911.
  52. Streit, W. and Entcheva, P. 2003. Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production. Applied Microbiology and Biotechnology 61: 21–31.
  53. Tylka, G. and Mullaney, M. 2015. Soybean cyst nematode-resistant soybeans for Iowa. Extension publication PM 1649, Iowa State University Extension, Ames, IA.
  54. Waldrop, G. L. , Holden, H. M. and Maurice, M. S. 2012. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms. Protein Science 21: 1597–1619.
  55. Wrather, A. , Shannon, G. , Balardin, R. , Carregal, L. , Escobar, R. , Gupta, G. , Ma, Z. , Morel, W. , Ploper, D. and Tenuta, A. 2010. Effect of diseases on soybean yield in the top eight producing countries in 2006. Plant Health Progress 10: 1094.
  56. Zempleni, J. 2005. Uptake, localization, and noncarboxylase roles of biotin. Annual Review of Nutrition 25: 175–196.
  57. Zempleni, J. , Hassan, Y. I. and Wijeratne, S. S. 2008. Biotin and biotinidase deficiency. Expert Review of Endocrinology & Metabolism 3: 715–724.

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