Soybeans are a great source of plant-based protein and produce considerable more protein per area of land than most other types of protein farming.
In the United States, one of the more damaging types of pest to soybean production is the soybean cyst nematode (SCN) and although there are some varieties of soybean that show some resistance to certain races of the nematode (currently the best known method of control), no variety is completely resistant and the number of known races of the SCNs mean that the threat of crop damage is constant and evolving.
With this in mind, a group of researchers from Tennessee (with funding from Tennessee Soybean Promotion Board) sought to build on previous research that had identified quantitative trait loci (QTL) associated with resistance to a variety of nematode races.
The article (from Plant Biotechnology Journal, which has been open-sourced by Wiley to its credit), cites previous research which identified a number of genes within the QTL that have been linked with resistance to some races of nematode. One in particular, the salicylic acid methyl transferase gene (GmSAMT1), had been previously studied by these researchers when they overexpressed the gene in soybean hairy roots and found that SCN race 2 development was significantly reduced. The GmSAMT1 gene plays a part in the production of salicylic acid (SA), a plant hormone that signals plant defence responses.
Developing this line of research further, the authors of the paper aimed to produce a number of generations of soybeans that overexpressed the GmSAMT1 gene to obtain further data on the possible correlation between the overexpressed gene and SCN resistance as well as looking at the effect of the overexpression on the plant’s agronomic traits.
The researchers cloned the gene coupled with a promoter along with a herbicide resistance gene into a binary vector which was inserted into the soybean variety ‘Williams 82’ by Agrobacterium tumefacians. Transgenic lines confirmed to contain the DNA insert were self-pollinated to create a number of lines of third generation (T3) soybean plants. The level of expression of the GmSAMT1 gene was ascertained using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR – a method turning specific messenger RNA into DNA which can then be counted). A number of lines demonstrated higher than control levels of transcription of the gene with one line of crops increasing GmSART1 transcription 226 fold compared to the control.
The transgenic lines were then inoculated with SCN. Lines were planted in two greenhouses, one of which was 5°C warmer than the other, and one lot planted in the field. The plants were left to grow for 35 days before being dug up and the number of cysts present were counted.
Transgenic leaf tissue taken after the 35 day inoculation period was used to perform a metabolite analysis of SA, benzoic acid (BA) and phenylalanine (PA) levels in the tissues for comparison to the control plant.
As described above, the expression of the GmSAMT1 gene was ascertained for a number of lines of transgenic plants and the control plants. The expression of inserted gene was significantly higher in the transgenic lines (to varying degrees) compared to the control.
Figure from article. Expression of the endogenous (figure a), inserted (figure b) and total expression (figure c) of the GmSAMT1 gene.
When the researchers looked at the infection of the plants with SCN they noted that the number of SCN eggs per cyst were not significantly different between the control and the transgenic plants, so they then counted the number of cysts on each plant to quantify the levels of infection. They found that there was a range of resistance in the transgenic lines with statistically significant reductions of infection with races 2, 3 and 5.
Correlating the total GmSAMT1 expression levels with the reduced levels of infection, a statistically significant negative correlation existed between gene expression and infection with SCN race 3, but not with the other races of nematode.
Figure from article. SCN infection levels of races 2, 3 and 5 on control and transgenic soybean lines.
They then report their findings on the agronomic effect of the gene overexpression, finding that the transgenic lines were all significantly taller than the control soybean. They also found no significant difference in seed yield or in single seed weight. Interestingly, they found that the lines grown in the warmer greenhouse tended to have a higher seed weight per plant and in two transgenic lines the seed number per plant was higher than the control. The field experiment showed no significant differences in agronomic traits and there were no noticeable differences in plant phenotypes.
Finally, the researchers analysed the metabolites within the plant tissue using mass spectrometry. What they found was that SA and BA levels dropped in correlation with GmSAMT1 expression while PA levels increased in a statistically significant manner. They expressed an opinion on why this might be so in the discussion of their results.
Many of the results speak for themselves so some of the discussion reiterated the correlations between transgenic plants containing the overexpressing GmSAMT1 gene and rate of infection of SCN found in the results.
The unexpected finding that the researchers point out was the non-linear correlation between overexpression and repression of different races of SCN. The transgenic line with the largest increase in gene expression had a significant correlation with reduced infection of SCN race three, while lines with lower expression of the gene had a higher resistance to races 2 and 5.
The researchers also point out the reduced BA and SA levels. They hypothesise that that a feedback look initiated by the reduced SA levels may have in turn reduced the levels of its precursor, BA, although why this occurred is not guessed at. PA, another important compound in SA biosynthesis was increased, was hypothesised to have been increased by the chorismate mutase enzyme, an enzyme that has previously been found to be secreted by numerous nematodes into plant tissue to manipulate the SA pathway. The precise explanation as to why PA levels affected by the nematode chorismate mutase enzyme in combination with the overexpressed gene would assist instead of hinder resistance isn’t given and is probably an area for future research.
In relation to plant growth and the noticeable increase in plant height and the increases in seed yield and number of seeds per plant in the warmer greenhouse, it is possible that increasing height is a strategy employed by the plant to overcome the deleterious effects of infection, while heat stress caused by the warmer greenhouse may induce a SA pathway response (previously been linked in other studies) which was increased in the case of the overexpressed GmSAMT1 lines.
What to take away
Taking one gene from QTL associated with a phenotypic trait is unlikely to yield broad spectrum resistance, and we don’t believe that the researchers expected such an outcome. What the research did do was correlate this particular gene and an increase in its expression with differing levels of resistance to a number SCN races, providing a useful building block to examine how genes within the other QTLs may help broaden the resistance. Further, it demonstrated that increasing resistance wouldn’t be done at the cost of the agronomic qualities of the plant.
What would have been interesting to see (although it may already be known – if it is, it doesn’t seem to be referred to in the paper), is what the metabolite levels were in a transgenic plant that hadn’t been infected with the SCN for comparison with infected transgenics and the control. From our reading of the methods, the mass spec analysis of metabolites was performed after the 35 day treatment with SCN and the data accumulated at that point. Given what we know about the secretion of enzymes into the plant by SCN and their effects on the SA pathway, identifying differences in metabolite levels between an infected and uninfected line may give a greater insight into any adapted response of SCN to the overexpressed gene.
Really promising research that gives some ideas of what further research is needed to gain the upper hand in this plant vs. nematode battle.