Role of phytohormones in pea-rhizobia and pea-agrobacteria interactions

 Phytohormones play a crucial role in all plant developmental processes. The phenotypic response to rhizobia (nodule absence/presence; their number and shape) and to agrobacteria (tumor and/or hairy root formation) are not exceptions. In our laboratory we use two approaches to study the involvement of phytohormones in plant-microbe interactions. The first is to study of the phytohormonal background of genotypes defective in phenotypical response to agrobacteria and rhizobia. The second is to investigate the characteristics of relationships with bacteria of known hormonal mutants. Our model organism is garden pea (Pisum sativum L.), and we presently have investigated more than 15 pea genotypes including lines differing in their geographical origin, symbiotic traits and morphological habit.

In in vitro experiments (transformation procedure and experiments with exogenous hormones) we used the stem explants of two-weeks-old aseptically grown plants. Strains of Agrobacterium were kindly provided by Dr. M. Ondrej (Institute of Molecular Biology, Academy of Sciences of the Czech Republic, Ceske Budejovice). Seeds of pea mutants and strains of Rhizobium were kindly provided by V. Tsyganov and A. Borisov (ARRIAM, Russia). Auxins (IAA - indole acetic acid; ILA - indole lactic acid; ICA - indole carbonic acid; IAld - indole acetaldehyde) were analysed using HPLC assay. Cytokinins (Z - zeatin; ZR - zeatin riboside; DZ - dihydrozeatin; DZR - dihydrozeatin riboside) were analysed by both HPLC and immunological assays.

Initally, we studied the ability of five pea lines to be transformed by wild type Agrobacterium tumefaciens (A281) and A. rhizogenes (8196) strains. All lines formed tumors in response to strain A281 and hairy roots in response to strain 8196. However, lines differed in their percentage of tumor/root formation, tumor size/root number and color. Some lines responded atypically to agrotransformation: line 32 developed both tumors and roots in response to A281, and when inoculated with strain 8796, lines 32 and 15 not only formed roots but also developed tumors. We compared the endogenous hormone contents of the lines investigated with their characteristics of reaction to transformation (Table 1). One can see that lines 4 and 9, which gave a typical response to agrobacteria, have an almost identical array of phytohormones. Lines 15 and 32, which exhibited unusual responses to agrobacteria, differ greatly from lines 4 and 9 and from each other. Therefore, unusual responses to agrobacteria may be related to alterations in hormonal composition, suggesting that reaction to agrotransformation really depends on endogenous plant hormone balance.

 Table 1. Phenotypic response of pea lines to the agrotransformation as related to their hormones composition and content

1mg/g of fresh weight; ²presence (+) or absence (-) is indicated.

Secondly we compared the characteristics of phytohormonal status between different nodule-forming mutants and their wild-type parents. We found: i) all mutants showed similar sensitivity to exogenous cytokinin as the parental lines; ii) both supernodulators (nod3, RisFixC) were more sensitive to exogenous auxin than their parents (cvs. Rondo and Finale respectively); iii) non-nodulating mutant E2 (sym5) also was more sensitive to auxin than Sparkle, whereas Nod- line R25 (sym8) showed the same reaction to applied auxin as the Sparkle parent; iv) comparison of endogenous auxin content in mutant-parent pairs revealed an increased auxin level in the aseptic seedlings the RisFixC mutant and the absence of differences between Sparkle and line E2. Our results showed, that early stages of pea-rhizobia interaction (e.g. curling of root hairs controlled by Sym8) appear to be independent of plant hormones, whereas nodule meristem initiation, determined by Sym5, and the plant's ability to regulate nodule number are potentially related with phytohormones and especially auxins.

Thirdly, we studied the involvement of phytohormones in control of plant interaction with microbes by using of the new pea hormonal mutant SGEcrt. This monogenic recessive mutant, having a modified root structure (curly roots), was obtained after EMS-mutagenesis of SGE line. We report that it has a two-fold increase in its auxin content, as well as a higher auxin sensitivity, over the SGE line. Thus, we believe SGEcrt is a pea auxin mutant.

Comparison of the reaction of SGEcrt mutant and the SGE line to A. rhizogenes inoculation revealed obvious differences between genotypes. Line SGE was susceptible to agrobacteria inoculation, it develops vigorous hairy roots and rare tumors, whereas line SGEcrt was absolutely insensitive to agrotransformation: no tumors and only one root were observed on its explants. There were differences between SGE and SGEcrt also in the case of interactions with Rhizobium leguminosarum bv. viceae (strain 250a) (Table 2). We suggest that the revealed peculiarities in reaction to agrotransformation and symbiotic parameters of crt mutant related with its changed auxin status.

Table 2. Comparison of the symbiotic characteristics of SGEcrt and SGE lines.

We conclude that pea genotypes with peculiarities in interaction with agrobacteria and rhizobia typically show altered phytohormonal phenotypes than wild type pea. And vice versa - a hormonal mutant can also be distinguished from its wild type parent by changed symbiotic and agrotransformation characteristics. Therefore, manipulations with plant hormonal traits can be used as a tool to change the efficacy of interactions with microbes.