A gene expression analysis of syncytia laser microdissected from the roots of the Glycine max (soybean) genotype PI 548402 (Peking) undergoing a resistant reaction after infection by Heterodera glycines (soybean cyst nematode)

Abstract

The syncytium is a nurse cell formed within the roots of Glycine max by the plant parasitic nematode Heterodera glycines. Its development and maintenance are essential for nematode survival. The syncytium appears to undergo two developmental phases during its maturation into a functional nurse cell. The first phase is a parasitism phase where the nematode establishes the molecular circuitry that during the second phase ensures a compatible interaction with the plant cell. The cytological features of syncytia undergoing susceptible or resistant reactions appear the same during the parasitism phase. Depending on the outcome of any defense response, the second phase is a period of syncytium maintenance (susceptible reaction) or failure (resistant reaction). In the analyses presented here, the localized gene expression occurring at the syncytium during the resistant reaction was studied. This was accomplished by isolating syncytial cells from Glycine max genotype Peking (PI 548402) by laser capture microdissection. Microarray analyses using the Affymetrix soybean GeneChip directly compared Peking syncytia undergoing a resistant reaction to those undergoing a susceptible reaction during the parasitism phase of the resistant reaction. Those analyses revealed lipoxygenase-9 and lipoxygenase-4 as the most highly induced genes in the resistant reaction. The analysis also identified induced levels of components of the phenylpropanoid pathway. These genes included phenylalanine ammonia lyase, chalcone isomerase, isoflavone reductase, cinnamoyl-CoA reductase and caffeic acid O-methyltransferase. The presence of induced levels of these genes implies the importance of jasmonic acid and phenylpropanoid signaling pathways locally at the site of the syncytium during the resistance phase of the resistant reaction. The analysis also identified highly induced levels of four S-adenosylmethionine synthetase genes, the EARLY-RESPONSIVE TO DEHYDRATION 2 gene and the 14-3-3 gene known as GENERAL REGULATORY FACTOR 2. Subsequent analyses studied microdissected syncytial cells at 3, 6 and 9 days post infection (dpi) during the course of the resistant reaction, resulting in the identification of signature gene expression profiles at each time point in a single G. max genotype, Peking.

The heterologous expression of aGlycine maxhomolog of NONEXPRESSOR OF PR1 (NPR1) and α-hydroxynitrile glucosidase suppresses parasitism by the root pathogenMeloidogyne incognitainGossypium hirsutum

“…These experiments prove that the method works to identify candidate genes that function in G. max defense to H. glycines parasitism Matthews et al 2013Matthews et al , 2014Pant et al 2014, Pant, Krishnavajhala et al 2015. In brief, G. max [Peking/PI 548402] and G. max [PI 88788] are infected with H. glycines [NL1-Rhg/HG-type 7/race 3] , resulting in a resistant reaction as proven histologically in unengineered roots which is the natural resistance response found in these G. max genotypes (Ross 1958;Endo 1965Endo , 1991Klink et al 2007Klink et al , 2009Klink et al , 2011Klink, Hosseini et al 2010;Klink, Overall et al 2010). Roots are processed for histology and laser microdissection (LM), a procedure that has been used to collect syncytia undergoing the defense response (Klink et al 2005(Klink et al , 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010).…”

“…In brief, G. max [Peking/PI 548402] and G. max [PI 88788] are infected with H. glycines [NL1-Rhg/HG-type 7/race 3] , resulting in a resistant reaction as proven histologically in unengineered roots which is the natural resistance response found in these G. max genotypes (Ross 1958;Endo 1965Endo , 1991Klink et al 2007Klink et al , 2009Klink et al , 2011Klink, Hosseini et al 2010;Klink, Overall et al 2010). Roots are processed for histology and laser microdissection (LM), a procedure that has been used to collect syncytia undergoing the defense response (Klink et al 2005(Klink et al , 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010). These procedures allow for the isolation of mRNA that then is converted to probe for hybridization onto the Affymetrix® Soybean GeneChip® (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010;Matsye et al 2011).…”

“…Roots are processed for histology and laser microdissection (LM), a procedure that has been used to collect syncytia undergoing the defense response (Klink et al 2005(Klink et al , 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010). These procedures allow for the isolation of mRNA that then is converted to probe for hybridization onto the Affymetrix® Soybean GeneChip® (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010;Matsye et al 2011). The hybridizations are run in triplicate (arrays 1-3) using a probe derived from RNA isolated from LM-collected syncytia obtained from three independent replicate experiments each run independently in the two different H. glycines-resistant genotypes (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010).…”

“…These procedures allow for the isolation of mRNA that then is converted to probe for hybridization onto the Affymetrix® Soybean GeneChip® (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010;Matsye et al 2011). The hybridizations are run in triplicate (arrays 1-3) using a probe derived from RNA isolated from LM-collected syncytia obtained from three independent replicate experiments each run independently in the two different H. glycines-resistant genotypes (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010). For the gene to be considered expressed at a given time point in the analysis presented here, probe signal is measurable above threshold on at least half of the arrays (three of six total arrays), combining both G. max [Peking/PI 548402] and G. max [PI 88788] (six total arrays), p < .05 (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010).…”

“…The hybridizations are run in triplicate (arrays 1-3) using a probe derived from RNA isolated from LM-collected syncytia obtained from three independent replicate experiments each run independently in the two different H. glycines-resistant genotypes (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010). For the gene to be considered expressed at a given time point in the analysis presented here, probe signal is measurable above threshold on at least half of the arrays (three of six total arrays), combining both G. max [Peking/PI 548402] and G. max [PI 88788] (six total arrays), p < .05 (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010). The original analysis procedure is performed as follows: the measurement for a particular probe set (gene) transcript on a single array is determined using the Bioconductor implementation of the standard Affymetrix®detection call methodology (DCM) (Klink et al 2007(Klink et al , 2009Klink, Hosseini et al 2010;Klink, Overall et al 2010).…”