ОЦЕНКА ГОМОЛОГИИ HOG1, 14-3-3 И STE11 У ПРЕДСТАВИТЕЛЕЙ СИМБИОТИЧЕСКИХ МИКОРИЗНЫХ ГРИБОВ

Climate change leads to desertification and land degradation, which leads to in biodiversity of woody and shrubby plants decrease

Drought tolerance increase was shown for plants Glycine max L., but species diversity of mycorrizal species was not studied in that research

Proteins Hog1, 14-3-3, Ste11 are playing important role in drought adaptation mechanisms launching in arbuscular mycorriza fungi in symbiotic bond “fungus-plant”. Hog1 and Ste11 are part of kinase cascade inhibiting large amount of genes, associated with drought tolerance

Regarding this, the goal of our research was to estimate amino-acid sequence homology of signal proteins Hog1, 14-3-3 and Ste11 amongst different mycorrizal fungi species for further study of symbiotic bond “fungus-plant” formation features in conditions of drought.

Signal proteins Hog1, 14-3-3, Ste11 presented among diverse species of arbuscular mycorriza were selected for research. Amino-acid sequences for alignment building were taken from Protein (NCBI) database and search for homologous of Hog1, 14-3-3 и Ste11 was carried out using program BLASAT. Protein sequences of Rhizophaus irregular, which genes are annotated and localised in chromosome, were selected as initial query. Homologous annotated as Hog1 were selected using instrument ProteinBLAST

1) Rhizophaus irregularis (XP_025185210.1);

2) Rhizophaus diaphanus (RGB42528.1);

3) Rhizophaus clarus (GBB87021.1);

4) Funneliformis geosporum (CAI2181536.1);

5) Glomus cerebriforme (RIA92778.1);

6) Geosyphon pyriformis (KAG9291825.1);

7) Ambispora gerdemannii (CAG8436886.1);

8) Diversispora epigaea (RHZ50009.1);

9) Acaulospora morrowiae (CAG8446886.1);

10) Racocetra persica (CAG8495386.1);

11) Cetraspora pellucida (CAG8501949.1);

12) Dentiscutata erythropus (CAG8739878.1);

13) Umbelopsis sp. PIM 123. (KAH8549606.1).

Proteins 14-3-3 and Ste11 were selected according to the same principle, but with slight changes in the species of the presented proteins, which are associated with the presence or absence of characterized amino acid sequences in the NCBI Protein databases

1) Rhizophagus irregularis DAOM (XP 025173882.1);

2) Rhizophaus irregularis (CAJ16742.1, PKY38380.1);

3) Glomus cerebriforme (RIA97653.1);

4) Gigaspora rosea (RIB16586.1);

5) Gigaspora margarita (KAF0484528.1);

6) Gigaspora rosea (RIB2229.1);

7) Glomus cerebriforme (RIA79980.1);

8) Gigaspora margarita (KAF0420307.1);

9) Funneliformis caledonium (CAG8442663.1);

10) Funneliformis geosporum (CAI2173046.1);

11) Acaulospora morrowiae (CAG8439107.1);

12) Cetraspora pellucida (CAG8612119.1);

13) Diversispora Epigaea (RHZ47341.1);

14) Ambispora gerdemannii (CAG8474580.1);

15) Geosiphon pyriformis (KAG9289207.1);

16) Dentiscutata erythropus (CAG8685133.1);

17) Racocetra persica (CAG8610165.1);

18) Umbelopsis sp PMI 123 (KAH8548527.1).

To search for the closest Ste11 homologues, the following amino acid sequences were taken from mycorrhizal symbiotic fungi:

1) Rhizophagus irregularis DAOM (XP 025186107.1);

2) Rhizophaus irregularis (XP 025172837.1);

3) Rhizophagus diaphanus (RGB34169.1, RGB38268.1);

4) Rhizophagus clarus (GES82803, GBB91729.1);

5) Funneliformis caledonium (CAG8474800.1, CAG8456822.1);

6) Funneliformis geosporum (CAI2186005.1);

7) Acaulospora morrowiae (CAG8664532.1, CAG8489781.1);

8) Glomus cerebriforme (RIA89482.1);

9) Dentiscutata erythropus (CAG8619186.1);

10) Gigaspora rosea (RIB01303.1);

11) Gigaspora margarita (CAG8472639.1);

12) Geosiphon pyriformis (KAG9304061.1);

13) Ambispora leptoticha (CAG8599465.1);

14) Diversispora Epigaea (RHZ44604.1);

15) Racocetra fulgida (CAG8484893.1);

16) Umbelopsis sp PMI 123 (CAG8583455.1, KAH8553767.1).

To identify differences between signaling proteins, bioinformatic analysis was performed. It included multiple alignment of the amino acid sequences of the studied proteins using the Mega 11 program (MEGA, Japan)

The Hog1 alignment showed that this protein is highly conserved. The greatest distance was between Umbelopsis sp. PMI 123 and Funneliformis geosporum: 0.18947. Umbelopsis is the most distant taxon from the rest, it belongs to the subphylum Mucoromycotina, while all other species belong to the subphylum Glomeromycotina. Among the subphylum Glomeromycotina, the greatest distance was found between Acaulospora morrowiae and Dentiscutata erythropus: 0.11892 No significantly variable regions were found throughout the amino acid sequence (Fig. 1), with the exception of the C-terminal region, where the length of the variable sequence is up to 50 amino acids (Fig. 2).

Figure 1 - N-terminus of Hog1 alignment

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Figure 2 - C-terminal variable regions of Hog1 proteins

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No major insertions or deletions were noted in the central region, with the exception of the missing first 27 amino acids in Acaulospora morrowiae and insertions at the C-terminus in Funneliformis geosporum. Hog1 phylogenetic tree, in general, repeats species phylogenetic structure. (Fig. 3).

Figure 3 - Hog 1 proteins phylogenetic tree

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The most distant protein from the rest is Umbelopsis sp. PIM 123, which belongs to a different subphylum. Its branch is much longer than all the others. The proteins Dentisculata Cetraspora and Racocetra, belonging to the family Gigasporaceae of the order Diversisporales, are classified into a separate clade. All proteins of species of the order Glomerales also form a separate clade. The branch lengths of Rhizophaus irregularis and Rhizophaus diaphanus are zero, and indeed these proteins have no substitutions, but Rhizophaus irregularis has several additional amino acids at the C-terminus of the chain.

The clade consisting of the proteins Geosyphon pyriformis, Ambispora gerdemannii and Acaulospora morrowiae differs from the phylogenetic tree of organisms, Ambispora and Geosyphon belong to the order Archaeosporales, and Acaulospora belongs to the order Diversisporales and the family Acaulosporaceae.

14-3-3 protein is also conserved, featured by a small variable region at the N-terminus, as well as a longer sequence in Cetraspora pellucida, far beyond the general conserved part of the protein at the N-terminus. (Fig. 4)

Figure 4 - N-terminal regions of 14-3-3 proteins

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No insertions or deletions in the internal part of the sequence were found in any species, but on the variable C-terminus additional amino acid sequences are present in Funneliformis geosporum and Racocetra persica. (Fig. 5)

Figure 5 - Variable C-terminal regions of 14-3-3 protein

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In the case of 14-3-3, two proteins were taken from some species, characterized as 14-3-3, but, nevertheless, the distances between these proteins are greater than between homologues from other species. For example, for Rhizophaus irregularis the distance between two 14-3-3 is 0.286386, and between 14-3-3 Rhizophaus irregularis (XP_025173882.1) and its closest homologue in Gigaspora rosea is 0.01988. Accordingly, the proteins present in two copies form two different clades, and the clade including 14-3-3 Rhizophaus irregularis PKY38380.1 diverges even earlier than the Umbelopsis sp PIM 123, indicating a very early divergence of 14-3-3 homologs, earlier than the divergence of subphylums. Glomeromycotina and Mucoromycotina (Fig. 6).

Figure 6 - 14-3-3 proteins phylogenetic tree

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The Ste11 protein is the least conserved of those presented in this article. It has variable regions not only at the ends (Fig. 7), but also in the central part of the sequence (Fig. 8).

Figure 7 - Variable N-terminus of Ste11 proteins

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Figure 8 - Insertions and variable regions in the internal part of Ste11 proteins amino-acid sequence

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Also in the internal part of the sequence there are long insertions in the protein sequences of Umbelopsis sp. PMI 123, Diversispora epigaea, Acaulospora morrowiae, Funneliformis caledonium, Ambispora leptoticha. Many species also have two Ste11 genes in their genomes, and, as in the case of 14-3-3, the distance between them is greater than between the closest homologues from other species, for example, the distance between Rhizophagus irregularis XP_025186107.1 and Rhizophagus irregularis XP_025172837.1 is 0.30817, while the distance between Rhizophagus irregularis XP_025186107.1 and Funneliformis caledonium CAG8456822.1 is 0.15729. But, unlike 14-3-3, all proteins of species of the family Glomeraceae converge into one clade, which indicates the divergence of these proteins after the formation of the family (Fig. 9).

Figure 9 - Ste11 proteins phylogenetic tree

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Both 14-3-3 and Hog1 proteins are highly conserved, making them unsuitable for use in phylogenetics. Phylogenetic analysis is also complicated by the presence in the genomes of fungi of several homologues of proteins of these groups, the differences in functions of which have not yet been studied. 

However, variability in the sequences of these proteins is present not only at the ends of the chain, which is typical for most proteins, but also in the central part. And although at the moment there is no data on the variability of the amino acid sequences of these proteins within species and the differences between different types of endomycorrhizal fungi in relation to the drought resistance factor, such data can be obtained in the framework of further studies, which leaves hope for linking variations in these proteins with the resistance of the “fungus-plan” pair to drought and its mechanisms.

Promoter regions of the genes of the studied proteins also require attention. Since the proteins themselves are highly conserved, markers for selecting symbiotic fungi as inocula should be sought in the promoter regions of their genes, attention to upstream and downstream regulators should be paid as well.

The Ste11 protein is much more variable; the distance to other proteins can reach 0.80988 (Umbelopsis and Diversispora epigaea); within Glomeromycotina, distances often exceed 0.4. This variability of the protein gives hope for detecting variations in its sequence associated with drought resistance of the “fungus-plant” system, which makes it a priority object for further research.

Alignments and phylogenetic trees were constructed for the Hog1, 14-3-3 and Ste11 proteins, which showed high conservativity of the Hog1 14-3-3 proteins and variability of the Ste11 protein. The divergence of the homologue pair 14-3-3 in Glomeromycotina before subphylum separation and Ste11 in Glomeraceae after the formation of the family was shown. Ste11 has been identified as a promising protein of interest for the search for intraspecific variations associated with resistance to drought and other abiotic stress factors.