Resistance genes in spruce trees raise hopes in struggle against pathogens

Increasing problems

Since the fungus is so efficient at colonizing new stumps and remains in the ground for so long, the prevalence of Heterobasidion in our forests increases with some 23 % each decade. Already, approximately one spruce tree in six is rotted to some extent, and the costs are estimated to 2 million SEK each day. Thus, there are lots of money to be saved – and even more to lose, if nothing is done.

Since it is difficult to target the fungus directly, rot resistant spruce trees appear to be the ideal counter measure. Such resistance is inheritable, which means a potential for breeding for resistance, but natural selection is a slow force in the case of Heterobasidion and spruce. The tree is rotting from the inside, but survives many decades, and the susceptible tree will thus keep regenerating again and again.

The quest for resistance genes

Selection could do well with some assistance. This might be achieved by artificial selection, in which only seeds from trees with coveted traits are used. However, resistance towards Heterobasidion is not noticeable until the tree is many years old. Scientists have long searched for molecular markers, identifiable parts of the tree DNA that can be associated with resistance, and thus can be used to point out resistant trees at a very early stage.

This quest has largely followed two principles. Genes active in spruce trees before and after pathogen infection have been identified, following the hypothesis that genes which control resistance are switched off or on in the presence of the fungus. Secondly, association studies have measured resistance in a large progeny of sibling trees, and analyzed what parts of the genome the strongest siblings have in common. Such parts are called QTL. Thus, one method show what genes are active during certain circumstances, but doesn’t prove their importance. The other show in what area the important genes are present, but it does not show whether they are actually active. A combination between these methods would be a potentially powerful method to identify resistance genes that could be used for breeding.

Combined methods

This is exactly what Rajiv Chaudhary, from the Department of Forest Mycology and Plant Pathology at the Swedish University of Agricultural Sciences, did in his recently published doctoral thesis.

– The aim of the thesis was to find molecular markers for resistance against Heterobasidion in spruce, and against ash decline in ash, Rajiv says. We wanted to combine the power of old and new association studies with new expression studies to highlight the issue from several angles at once.

Rajiv and his colleagues used a compilation of several past analyses of spruce sibling families, which gave a highly resolved image of what genes are present in the QTLs associated with resistance. Subsequently, bark inoculations with Heterobasidion were conducted on young spruce trees, in order to study which of these genes were actually affected by the infection.

Several strong candidates

– This approach resulted in 124 genes that were significantly affected by the infection and simultaneously present within a QTL. Some of these are known from previous studies as involved in resistance against infections, Rajiv says.

For example, PaNAC04 was strongly upregulated during infection. NAC is a transcription factor, which affects the expression of other genes. Many NAC-genes has been proved to be of importance in plants’ responses to biotic and abiotic stresses, and also to directly affect tolerance to intruding pathogens of various kinds.

Complementing studies

– Sibling families are useful, but they cannot find QTL for traits with no discernable difference between the parents, no matter how important they might be to resistance, Rajiv says. We complemented these with an independent association study, using 466 non-related spruce trees. The principle is akin to that of the sibling studies; if resistant individuals share sequences of the genome, it can be assumed that these sequences, or QTL, contain genes that affect resistance.

The association study caught the gene PaLAC5, significantly expressed in the bark of the infection wound, and only so during Heterobasidion infection. Previous studies have shown that PaLAC5 is activated during stress responses. It controls production of a protein called laccase, which is involved in the production of lignin. Lignin makes tissues inhospitable to the intruding fungus.

Conclusion

Rajiv Chaudharys thesis has identified a number of genes that can be used as markers for resistance in spruce. The spruce genes are of great interest to breeding work, since they have been verified with a combination of methods and, in some cases, also shown to be expressed in the actual infection zone.

Written by Mårten Lind.