Identification of sequences encoding for ncr-peptides and defensins in the ‘meta-assembly’ of transcriptome of pea (pisum sativum l.) Nitrogen-fixing nodules

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iDENTIFICATION OF SEQUENCES that ENCODe NCR PEPTIDES AND DEFENSINS IN THE “META-ASSEMBLY” OF PEA (PISUM SATIVUM L.) NITROGEN-FIXING NODULE TRANSCRIPTOMEs

Zorin Evgeny Andreevich

Kliukova Marina Sergeevna

Kulaeva Olga Alekseevna

Afonin Alexey Mikhailovich

Tikhonovich Igor Anatolievich

Zhukov Vladimir Aleksandrovich

 

Federal State Budget Scientific Institution “All-Russia Research Institute for Agricultural

BACKGROUND

Formulations that include antibiotics and insecticides of different natures are often used to maintain the yield of agricultural crops by protecting them against pathogenic microorganisms. However, many antibiotics can cause microbial resistance, which significantly reduces their effectiveness and calls for the development of new formulations [1, 2]. Thus, the search for naturally existing molecules that have antimicrobial activity is of great importance.

Plants, being fixed organisms, are unable to escape the effects of biotic and abiotic environmental factors and, instead, have to adapt to them. Therefore, interaction with different symbiotic microorganisms that enhance the adaptive potential of the plant–microbe system is very important for the plant lifecycle [3]. However, only some microorganisms are useful to plants, whereas others demonstrate pathogenic properties; therefore, in the course of evolution, plants have developed multi-faceted and various protection systems against microorganisms. A number of plant systems aim to control microbial life inside the plant, the main one being the immune response, which consists of identification and destruction of pathogenic bacteria and fungi. One component of this system is antimicrobial peptides (AMPs), also known as defensins [4]. Defensins are cysteine-rich peptides consisting of 45–54 amino acids. Defensins of plants, fungi, and mammals are similar in structure and function. Most defensins are involved in the inhibition of fungal infection. Some representatives of the family also demonstrate antibacterial activity and are involved in the development of resistance to some abiotic factors [5]. Defensins and other AMPs (for example, hevein-like peptides and thoinines) monitor microorganisms at the whole-plant level and are produced in all tissues and organs. In addition to whole-plant systems, local systems ensure response to bacterial penetration; for example, these local systems exist in the nitrogen-fixing nodules of leguminous plants attributed to inverted repeat­lacking clade (IRLC), presented by lucerne (Medicago truncatula Gaertn.), garden pea (Pisum sativum L.), horse beans (Vicia faba L.), creeping trefoil (Trifolium repens L.), etc. [6–9].

It is known that leguminous plants have a selective advantage in their ability to enter into symbiotic relationships with soil bacteria. Bacteria penetrate through the root hairs of plants into the special de novo formed organ, the root nodule, in which the processes of endosymbiont differentiation take place, including the conversion of bacteria into a symbiotic form known as bacteroides [10]. This process results in the bacterial conversion (fixation) of atmospheric nitrogen into a form that is available to the host plant [11]. Recently, it has been demonstrated that the process of terminal (irreversible) differentiation of endosymbionts in IRLC leguminous plants is controlled by a family of defensin-like nodule-specific cysteine-rich (NCR) peptides, which are probably also a part of the immune system of the nodule [7]. It is supposed that NCR peptides, as well as defensins, perform their biological functions by means of demonstration of antimicrobial activity [12–16].

Defensins and NCR peptides vary widely in their nucleotide and amino acid sequences, which allows them to suppress the vital activity of a wide range of microorganisms and do not cause resistance [17]. Because of the growing issue of resistance to antibiotics, the search for and study of these molecules in different plants, both model and non-model, is an important area of research.

In pea plants, in contrast to the truncated medick plant model, the search for representatives of the family is complicated because of the absence of genome sequencing data. On the other hand, there is a considerable amount of data on the available transcriptome databases for the organs and tissues of Pisum sativum, including the nodules. However, these transcriptome data were assembled by different methods (assembly programs) with different parameters, which has resulted in the loss of some information. In order to obtain more detailed information about pea transcripts, we created a meta-assembly of the nodule transcriptome of P. sativum by using published readings of transcriptomes of different pea lines. This meta-assembly is a combination of separately assembled (by means of one assembly program with similar parameters) transcriptomes of pea nodules of different lines. Our analysis aims to improve the effectiveness of searches for target sequences in comparison with previous work, which used data generated by different work groups using different software tools. Our meta-assembly was successfully used for the search for AMPs attributed to the families of defensins and NCR peptides.

MATERIALS AND METHODS

At the first stage of work, the search for peptides with antimicrobial activity was conducted by means of existing assemblies of transcriptomes of pea nodules available in the NCBI database. Assemblies of the lines Kaspa, Parafield, Cameor, and SGE were used for the work [18–20].

The second stage involved improvement of available assemblies by means of their re-assembly using the latest, updated version of the assembly program Trinity and compilation into meta-assembly.

The quality of readings was controlled by means of FastQC [21]. Readings of low quality and adapters were deleted to BBduck from the BBmap package [22]. After filtration of the sequencing data of the pea line, Cameor, nodules, 148,408,730 (89%) readings were left out of 166,803,965 readings; for SGE, 42,054,133 readings out of 52,021,865 readings were left (81%); for Kaspa, 30,945,747 out of 31,256,637 readings were left (99%); and for Parafield, 20,637,281 out of 20,842,187 readings were left (99%). The assembly of transcriptomes out of the readings that passed processing was made by means of the program Trinity (ver. 2.8.4) [23]. The quality of assemblies was assessed by means of transRate [24] (Table 1).

Contig structural annotation was conducted by means of Transdecoder [25]. Assembly of transcriptomes for quantitative analysis (quantification) and analysis of single-nucleotide polymorphisms included removal of transcripts without the reading frame or with the reading frame (when less than 100 nucleotides), removal of duplicated transcripts and sequences overlapping for 50% and more with 100% similarity, and removal of all contigs, for which homologous sequences were not found in the available databases. However, the goal of the work was to search for short AMPs, and the abovementioned operations can result in the loss of part of the target sequences, which is crucial in our case. That is why only 100% identical sequences were deleted (397 transcripts, 0.09% of the total), and all contigs containing reading frames longer than 90 nucleotides were retained. Further analysis was conducted on transcriptomic assembly of the pea nodules of different lines, which included 349,953 contigs.

Search of AMPs in the de novo built meta-assembly of transcriptomes of the pea nodules was conducted using the SPADA program [26], a pipeline that arranges the start sequences using the tools Augustus [27], Glimmer [28], GeneWise [29], and SignalP [30]. Detection of signal sequence in the detected peptides and separation of signal peptides from the mature section were performed using the SignalP program. The presence of four or six cysteines in the mature section of the NCR peptide was validated by means of multiple alignment using the program MAFFT [31] with option G­INS­i for more accurate alignment. The physical and chemical properties of the mature peptides were forecasted using the service DBAASP [32]. The service work results were processed by means of a script made in PHP language. The antibacterial activity of detected peptides was forecasted by means of the web service CAMP3 [33]. Results were analyzed by means of script made in Python language.

RESULTS

The first round of searches for sequences coding NCR peptides in already-obtained transcriptomic layouts of the pea nodules allowed identification of 553 de novo NCR peptides. 355 additional sequences not detected in the previous layouts were identified as a result of transcriptome re-layout and repeated search using SPADA. Thus, 908 unique sequences coding NCR peptides were detected. The boundary between the signal sequence and the mature section of the peptide was forecasted in 838 of these sequences, according to which only forecasted sequences of mature peptides were used for further analysis.

Physical and chemical properties and antibacterial activity were forecasted for the mature section of the 838 NCR peptides using computer-aided learning methods (Tables 2 and 4). Additionally, 55 defensins were identified, 41 of which had a signal sequence and junction site between the signal and mature sequences. The results of the forecast of antibacterial activity for 41 defensins are provided in Tables 3 and 5.

DISCUSSION

A number of unique mechanisms aimed at protection against pathogens has been formed in plants during the process of evolution. These mechanisms include physical barriers and a wide range of synthesized secondary metabolites and AMPs. One of the largest families of AMPs is the defensins, which are attributed to evolutionary ancient sequences that occur in all major eukaryotic organisms [5]. Except for conservative residues of cysteine, these peptides demonstrate very low similarity of sequence, which explains the wide range of activities (antifungal, antibacterial, and activation by abiotic factors) demonstrated by these peptides [5]. Besides defensins, several families of defensin-like sequences have been detected in plants, some of which have more specific natures of expression and more focused functionality. Thus, the group of NCR peptides detected in leguminous plants plays an important role in the differentiation of Bacteroides and the control of symbiotic development [34]. The goal of this study was to identify and characterize representatives of the family of defensins and NCR peptides in pea plants.

Optimization of the data processing algorithms, including re-layout of available pea transcriptomes using a unified method by means of the updated version of the program Trinity with their further combination, allowed detection of 908 sequences expressed in the nodules that code NCR peptides and 55 sequences that code defensins. It is recommended that the complications of meta-assembly of transcriptomes be reduced by eliminating duplicates, as well as transcripts that do not contain reading frames or contain reading frames that are shorter than 90 nucleotides, in order to save time and resources prior to searching for antibacterial peptides by means of SPADA. However, the set of peptides obtained in the search process shall be additionally checked by means of analysis of multiple alignment for the presence of depressed erroneous results, which are inevitable in the process of algorithm operation.

The role of defensins in the development of plant–microbe symbiotic interactions is poorly studied. Only a few studies demonstrate involvement of defensins in symbiosis. Comparative transcriptomic analysis of roots and nodules formed in the process of the development of symbiosis by the plant Datisca glomerata and by representatives of the genus Frankia allowed detection of a group of defensins expressed in the nodules in a special way that differ from the other defensins with the presence of a special terminal domain [35]. Nodule-specific expression of the number of genes encoding defensins was demonstrated in the research into Medicago truncatula [34]. Activation of expression of the number of genes coding defensin-like peptides was detected during interaction of Japanese bird's-foot (Lotus japonicus (Regel.) K. Larsen) with fungi of the arbuscular mycorrhiza, Rhizophagus irregularis [36]. However, the specific role of detected peptides in the development of symbioses is still unknown.

Despite the similarity with defensins, the availability of conservative cysteine pattern, high variability of sequences, possibility of occurrence of antimicrobial activity, which is probably explained by the kinship of these two gene families, the main function of NCR peptides is the differentiation of the non-symbiotic bacterium into the nitrogen-fixing bacteriodes within the symbiotic compartment [37–39]. It is known that NCR peptides, especially ones with a high positive charge, can interact with electronegative bacterial membranes, specifying formation of pores on the surface of bacterial cells. The strong effect of such peptides can result in bacterial cell lysis [16]. However, in case of symbionts, pore formation has a temporary nature, and pores are probably required for penetration of other NCR peptides, which interact with the intracellular targets that modify cell physiology and morphology [15, 40]. It is supposed that an important condition is the availability of protein BacA on the bacterial membrane, which also promotes NCR peptide import inside the cell, where they can be decontaminated by means of the bacterial system of protein degradation [41, 42].

In this study, 908 unique sequences coding NCR peptides were found in P. sativum. It should be noted that more than 700 representatives of the family have been described for truncated medick so far, which is not surprising as the pea genome is almost 10 times larger than the medick genome. On the other hand, chimeric sequences can present among the pea peptide sequences detected in the work because of imperfection of the algorithms of layout; besides, some detected sequences can be the allelic conditions of the same AMP-coding gene. The problem of chimeric sequences can be resolved only after whole-genome sequencing. The problem of allelic variants can be resolved by means of sequencing of the transcriptomes of each of the studied pea lines and assessment by reading every peptide. Thus, methods of next-generation sequencing shall be used for the verification of detected peptides and will be the subject of further research.

Representatives with forecasted antimicrobial activity were detected among defensins and NCR peptides. Larger amounts of sequences with a high level of probability of antimicrobial activity were detected among NCR peptides in comparison with the identified defensins, which indicates their better representation in the transcriptomic data. Such diversity of NCR peptides in terms of the content and, accordingly, the possibilities and levels of antimicrobial activity in the nodule cells can certify that NCR peptides inside the nodule functionally substitute defensins and form the local immune system of the plant, which eliminates undesirable bacteria and at the same time promotes differentiation of symbiotic partners.

CONCLUSION

Identification and characterization of defensins and defensin-like peptides are important for the understanding of their roles in plant protection responses, as well as for the detection of new antimicrobial compounds. This area of research is urgently required as pathogenic bacteria of agricultural plants inflict significant economic damage and affect food safety [43]. The interest in defensins is justified by the possibility of developing new types of protective agents on their basis. As AMPs can seriously vary in different types of plants, the important task is identification of AMP across many species, including agriculturally valuable crops. Leguminous plants are an interesting object for AMP study as they contain both defensins and defensin-like NCR peptides unique to leguminous plants. Because defensins and NCR peptides are numerous and variable in their content, attack different targets in the cells of pathogenic microorganisms, and are jointly produced, microorganisms cannot resist such a cocktail of AMP and obtain resistance to them. The identified AMPs with a wide range of activities can be used to counteract plant pathogens, as well as the pathogens of animals and humans.

Characterization of natural antimicrobial molecules is an important area of research. The example of protein families studied in this work and the description of these molecules contribute much to the understanding of the processes of interaction of plants and bacteria, as well as revealing the mechanisms of different responses to interaction with pathogenic and useful bacteria. Understanding the characteristics of antimicrobial activity allows the construction or modernization of existing peptides, which can be used as new-generation antibiotics, as well as managing the effectiveness of nitrogen-fixing symbiosis.

 Acknowledgment

The work of antimicrobial sequences detection attributed to NCR peptides family was supported by the Russian Foundation of Basic Research (Grant No. 18­34­00187 мол_а). Analysis of sequences coding defensins was financed by the State Task of the Rf Ministry of Education and Science (No. 0664­2019­0022­С‑01) for 2019.

 

Table 1

Description of transcriptomes of pea nodules collected within the frame of the work

 

Pea line	Contig average length	Percent of transcripts with reading frames	Total contigs
SGE	932.3	65.5	48 649
Cameor	877.7	47.4	212 147
Kaspa	487.1	62.7	113 512
Parafield	484.6	61.9	91 831

 

Table 2

Forecast of antimicrobial activity of NCR peptides' mature part

 

Quantity of peptides	Support vector machines	Random forest	Discriminatory analysis
With antimicrobial activity	328	422	332
Without antimicrobial activity	510	416	506

 

Table 3

Forecast of antimicrobial activity of defensins' mature part

 

Quantity of peptides	Support vector machines	Random forest	Discriminatory analysis
With antimicrobial activity	15	22	16
Without antimicrobial activity	26	18	25

 

Table 4

Amino acid sequences of five NCR peptides having the highest forecasted antimicrobial activity

 

Sequence	Support vector machines	Random forest	Discriminatory analysis
QACIHDRQCRCTQHTVSKCINGFCKCYISNT	0.937	0.893	0.994
IKCDVQADCPKIPNLFPAIYKCINKKCRLIG	0.951	0.923	0.995
IVVTVDIKEIFKCESHKQCRKQMPNCKRPKIARCVSRTCKCW	0.865	0.698	0.988
KRACTYHHQCSDISCSYGYISLCIEKYCHCVKN	0.987	0.860	0.994
KKIKCRTDSDCPKQMCRFPTYNKCVIRNHCKCVLSRII	0.891	0.751	0.990

 

Table 5

Amino acid sequences of five defensins having the highest forecasted antimicrobial activity

 

Sequence	Support vector machines	Random forest	Discriminatory analysis
KVCEKRSKTWSGFCGKTRNCKKQCINVENAVFGACHRQGFFGFACFCYFKC	0.984	0.967	0.998
KTCENLSGTFKGPCIPDGNCNKHCRNNEHLLSGRCRDDFRCWCTRNC	0.826	0.945	0.902
RLCGRTSKTWRGPCYINLSCNAECTMKEQAIFGTCQHFECFC	0.664	0.851	0.816
RDCESKSHKFKGTCLSDTNCASVCQTERFTGGHCRGFRHRCYCTTHC	0.923	0.845	0.917
KFCRSKSRTWSGACIDNSPCSTECKQLEHASHGACRHNGFCFCYFNC	0.913	0.935	0.962

 

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About the authors

Evgeny A. Zorin

Federal State Budget Scientific Institution “All-Russia Research Institute for  Agricultural Microbiology”

Author for correspondence.
Email: kjokkjok8@gmail.com

engineer-researcher, Laboratory of Genetics of Plant-microbe Interactions

Russian Federation, Saint-Petersburg, Podbelsky chausse, 3.

Marina S. Kliukova

Federal State Budget Scientific Institution “All-Russia Research Institute for Agricultural Microbiology”

Email: marina.kliukova@gmail.com

engineer-researcher, Laboratory of Genetics of Plant-microbe Interactions

Russian Federation, Saint-Petersburg, Podbelsky chausse, 3.

Olga A. Kulaeva

Federal State Budget Scientific Institution “All-Russia Research Institute for Agricultural Microbiology”

Email: okulaeva@arriam.ru

Phd, Laboratory of Genetics of Plant-microbe Interactions

Russian Federation, Saint-Petersburg, Podbelsky chausse, 3.

Alexey M. Afonin

Federal State Budget Scientific Institution “All-Russia Research Institute for Agricultural Microbiology”

Email: afoninalexeym@gmail.com

engineer-researcher, Laboratory of Genetics of Plant-microbe Interactions

Russian Federation, Saint-Petersburg, Podbelsky chausse, 3.

Igor A. Tikhonovich

Federal State Budget Scientific Institution “All-Russia Research Institute for Agricultural Microbiology”; Federal State Budget Educational institution of higher education "Saint Petersburg state University"

Email: arriam2008@yandex.ru

PI, academician of RAS, Sc. D., professor

Russian Federation, Saint-Petersburg, Podbelsky chausse, 3.

Vladimir A. Zhukov

Federal State Budget Scientific Institution “All-Russia Research Institute for Agricultural Microbiology”

Email: vzhukov@arriam.ru

Phd, head of laboratory, Laboratory of Genetics of Plant-microbe Interactions

Russian Federation, Saint-Petersburg, Podbelsky chausse, 3

References

Supplementary files