Functional Analysis of Calmodulin Binding Protein 60s in Disease Resistance Responses in Rice (Oryza Sativa L.)

Occurrence and prevention of diseases have a big impact on sustainability of staple food crop like rice. The crosstalk between phytohormones and secondary messengers in host cell during infestation of pathogens play a pivotal role in defence responses. Apart from SA and JA, the role of brassinosteroids (BR) in defence responses in plants is unprecedented. The calcium signatures observed during early infection process modulates the expression of calmodulin and other Ca 2+ binding protein followed by their interactions with calmodulin binding protein (CBP), which are pivotal in elucidating defence responses in plant. Numerous CBPs have been identied, which modulated stress responses with the help of CBD and other functional domain. Interestingly, Arabidopsis CBP60 (AtCBP60) family protein, SARD1, was involved in defense responses via SAR. However, no rice CBP60 (OsCBP60) has been identied in relation to pathogen infection yet.


Results
In present investigation, 15 OsCBP60 genes were identi ed using BLASTP searchers using AtCBP60s as bait sequences. Expression studies showed that 3 OsCBP60s (OsCBP60_5, OsCBP60_10, and OsCBP60_15) genes were upregulated consistently in all the time point studied in rice seedlings treated with fungal (Magnaporthe oryzae) and bacterial (Xanthomonas oryzae) pathogens. Differential expression of OsCBP60s genes were observed in salicylic acid (SA), epi-brassinosteroid (EBR) and jasmonic acid (JA) treated rice seedlings. Taken together, OsCBP60_5 was found to be upregulated in both pathogens and two phytohormones (SA and EBR) treatment.

Conclusions
The differential expression of OsCBP60s genes under phytohormones and pathogens treatment suggests that these genes might be important targets for increasing biotic stress responses in rice.

Background
Rice being a global staple food makes it a backbone for food and nutrition security in most of the South East Asian countries. The consumption on rice has increased tremendously and further projected to increase by 34% in 2050 due to ever increasing population (Romero and Andrés Gatica-Arias 2019).
However, the occurrence of several environmental stresses limits the yield production and causing huge losses in recent decades (Romero and Andrés Gatica-Arias 2019). The diseases of rice, which cause losses ranges from 12-15% (IRRI, 2016) are among the major bottleneck in maximizing the rice yield.
Around 70 diseases are reported to infect rice during its life cycle having varying mechanism of infestation (Saha et al., 2015). Losses caused due to bacterial blight (Xanthomonas oryzae pv. oryzae) and blast diseases (Magnaporthe oryzae) are most paramount (Fu et al., 2012). To cope up with these stresses, plants have evolved complex sophisticated mechanisms for sensing and transduction of stress stimuli to mount appropriate biological responses (Casal, 2002). Phytohormones and a variety of secondary messengers play key roles in mediating cellular responses to various stress stimuli (Bargmann and Munnik, 2006; Bari  The calcium ion (Ca 2+ ) is an ubiquitous and highly versatile secondary messenger, which regulates growth and development, as well as stress responses using speci c calcium signature in plant (Hetherington and Brownlee 2004;Yang and Poovaiah 2003 (Reddy et al., 2002). In silico domain prediction analysis of different members of AtCBPs showed conservation of the calmodulin binding domain (CBD) and other domains (Reddy et al., 2002).
Interestingly, AtCBP60 family contains only CBD domain. AtCBP60 family comprises 8 members, which regulates variety of stress responses stresses (Truman et al., 2013;Wan et al., 2012). Previous study showed production of SA in response to recognition of microbe-associated molecular patterns (MAMPs) were regulated by AtCBP60g, which further limits the growth of the bacterial pathogen Pseudomonas syringae pv. maculicola (Pma) (Wang et al., 2011). However, to best of our knowledge, no studies have been carried out in identi cation and characterization of OsCBP60s relation to defence responses in rice

Sequence analysis
Similarity searches on nucleotide and amino acid sequences were carried out using BLASTP at the National Center for Biotechnology Information (NCBI) GenBank database or Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) database. A phylogenetic tree was constructed using neighbour-joining (NJ) method based on the genetic distance of the protein sequences using MEGA 7 (http://www.megasoftware.net/) tool.
Plant materials and treatments.
Rice cv. Rajendra Kasturi (Oryza sativa L. sp. indica cv. Rajendra Kasturi) was used in this study. Seeds of Rajendra Kasturi were grown in earthen pots having soil: cocopit (2:1) and maintained in the greenhouse (28 and 23°C, day and night, respectively). M. oryzae (isolate B157, corresponding to international race IC 9) was obtained from Dr. Bharat Chattoo Genome Research Centre, M.S. University, Vadodara, Gujarat. M. oryzae were grown on Potato Dextrose Agar (PDA) medium, at 28-30ºC. For M. oryzae treatment, 21-dayold rice seedlings were inoculated with conidial suspensions (1x10 5 spores/ml) of M. oryzae as described previously (Bonman and Mackill 1988;Jha et al., 2008). The seedlings were grown in a greenhouse maintained at 28 o C. Rice seedling treated with distilled water without M. oryzae spores were act as a mock treatment.
X. oryzae pv. oryzae was isolated from blight infected rice eld at Bihar Agricultural College, Sabour (NCBI GenBank: MH986180). The culture of X. oryzae was grown on nutrient agar medium at 28-30 °C. Leaf infection with X. oryzae was performed using leaf clipping method as described previously (Kauffman et al., 1973). Rice leaves clipped with scissor dipped only in saline (0.9%) containing 0.05% Triton-X-100 were act as a mock treatment.
For SA treatments, 21 days old rice seedlings were sprayed with 3 mM sodium salicylate containing 0.05% Triton-X-100 (Prasad et al., 2009). The rice seedlings sprayed with distilled water containing 0.05% Triton-X-100 act as a mock treatment.
For JA treatment, 21 days old seedlings grown in black portrays (9 cm diameter and 9 cm height) containing small hole (1 cm diameter) at the bottom for water absorption from a tray (20 14 7 cm) containing 1 litre of water. Seedlings were then placed on another tray (20 14 7 cm) containing 100 mM jasmonic acid (Yamada et al., 2012). Seedlings placed on distilled water were acts as mock.
For EBR treatment, surfaced sterilized rice seeds were placed on ½ MS containing 1 µM EBR media in test tubes (Sahni et al., 2016). The test tubes were closed with sterilized cotton plug. The rice seeds grown on MS media containing 0.02% ethanol were acts as a mock. EBR treatment was given for 15 days and leaf tissues were collected for gene expression analysis.
Rice samples were used for Total RNA isolation using SV Total RNA isolation kit (Promega).The isolated total RNA samples were used for cDNA synthesis using random hexamer primers by following manufacture's protocol (Promega). cDNA were diluted in nucleases free water (1:5) and used for Quantitative real-time RT-PCR (qRT-PCR) analysis. qRT-PCR was carried out using SYBR Green dye on Light Cycler system (Applied Biosystem). Each qRT-PCR quanti cation was carried out in triplicate using primers for each individual gene (Table S1). The expression value of ACTIN was used to normalize the expression data of genes. The fold change in terms of gene expression level was calculated using 2 -∆∆Ct method of relative quanti cation compared with control (Livak and Schmittgen, 2001). The expression studied of 15 OsCBP60s along with OsPR1a gene (Table S2) in treated rice seedling were performed at 12h, 24h and 48h after pathogens and hormonal treatments.

Identi cation of rice OsCBP60s genes
The protein sequence of all the AtCBP60s genes (Reddy et al., 2002) were extracted using Phytozome11 (https://phytozome.jgi.doe.gov/pz/portal.html). BLASTP searches were performed using the complete protein sequence of each AtCBP60 protein separately as the query sequence and total list of OsCBP60s were prepared. The duplicate entries and apparent incomplete sequences were removed manually. Fifteen candidate OsCBP60s genes were obtained (Table S1). Further, protein sequences of 15 OsCBP60s and 8 AtCBP60s were used to create a phylogenetic tree using neighbor joining (N-J) tree with bootstrapping (500 reps) in MEGA7 software (http://www.megasoftware.net/) (Fig. 1). Two major clusters were obtained, which were further divided into different sub cluster as shown in Fig. 1. Majority of the AtCBP60s were grouped in cluster I except SARD1 and AtCBP60g. Both SARD1 and AtCBP60g were previously shown to be involved in systemic acquired resistance (SAR) in Arabidopsis (Zhang et al. 2010). However, OsCBP60s were dispersed in both the cluster. Interestingly, LOC_Os01g04280 (OsCBP60_14) and SARD1 (AtCB60h) were fell within the same clade.

Protein domain search
The Pfam analysis showed that like AtCBP60s all the identi ed OsCBP60s contains only one domain i.e. calmodulin binding protein (CBP) domain (Fig. 2). However, the size and position of CBP domain varies in Arabidopsis and rice CBP60s. The size of CBP domain varies from 283 to 346 aa.

Identi cation of transcription factor binding sits (TFBSs)
The upstream sequences (1000 bp) of OsCBP60s genes were analysed using PlantPan VERSION. 2/. Several TFBSs involved in stress responses were found to be enriched in the promoter sequence of OsCBP60s (Fig. S1). In total 1529 stress responsive TFBSs were observed in positive or negative strand of OsCBP60s ( Fig. 3 and 4).  Table S3). Oxidative stress responsive elements like ARE (AAACCA), ethylene-responsive ERE (ATTTTAAA), AREs (Anaerobic responsive elements), GC-motif (CCCCCG) presents in some of members of OsCBP60s genes with consequences TGACG (Table S3).

Expression pro ling of OsCBP60s after pathogens treatment
To gain further information on the biological function of OsCBP60s, Quantitative real-time reversetranscriptase (RT) polymerase chain reaction (qPCR) analysis were performed in rice seedlings treated with two most devastating rice pathogens (M. oryzae and X. oryzae) at 12, 24 and 48 h after treatment. In present investigation, differential expression of OsCBP60s was observed in rice seedlings treated with pathogens ( Fig. 5 and 6). OsPR1a was also found to be upregulated in all the time points studied after treatment with M. oryzae and X. oryzae (Fig. 5 and 6). The upregulation of OsPR1a in all the time point studied indicates onset of defense mechanism in rice against both the pathogens Treatment with M. oryzae leads to the up regulation of all OsCBP60s with different magnitude in all the time point studied. Five OsCBP60s (OsCBP60_5, OsCBP60_6, OsCBP60_7, OsCBP60_11 and OsCBP60_14) showed high level of upregulation at early time point i.e. 12 h. Interestingly, six OsCBP60s namely OsCBP60_4, OsCBP60_5, OsCBP60_9, OsCBP60_10, OsCBP60_12 and OsCBP60_15 showed consistently up regulated at 12 h, 24 h and 48 h (Fig. 5). Three OsCBP60s (OsCBP60_5, OsCBP60_6 and OsCBP60_11) showed high level of upregulation at 24 h after treatment with M. oryzae.
Treatment with X. oryzae caused differential expression of OsCBP60s (Fig. 6). qRT-PCR analysis showed that induced expression of 4 OsCBP60s (OsCBP60_6, OsCBP60_7, OsCBP60_8 and OsCBP60_9) occurred as early as 12 h after treatment with X. oryzae but downregulates at 24 h and 48 h. Interestingly four genes (OsCBP60_4, OsCBP60_5, OsCBP60_14 and OsCBP60_15) showed consistently up regulation at 12 h, 24 h and 48 h (Fig. 6) after treatment with X. oryzae. Taken together, OsCBP60_5, OsCBP60_10 and OsCBP60_15) were found to be commonly upregulated in M. oryzae and X. oryzae treatments.
Most studies have identi ed the antagonistic interactions between the SA and JA mediated signaling pathways (Takahashi et al., 2004). The down regulation of OsPR1a at 24 h and 48 h after JA treatment con rms the effectiveness of treatment. Five OsCBP60s (OsCBP60_1, OsCBP60_5, OsCBP60_7, OsCBP60_13, and OsCBP60_14) showed downregulation in all the time studied after JA treatment (Fig.  8). However, the expression of two OsCBP60s (OsCBP60_4 and OsCBP60_15) were found to be upregulated at 12 h followed by downregulation at 24 h and 48 h of JA treatment (Fig. 8). Interestingly, 4 OsCBP60s (OsCBP60_7, OsCBP60_8, OsCBP60_9 and OsCBP60_12) were upregulated at all the time point studied after JA treatment (Fig. 8).
In EBR treated rice samples all the 15 OsCBP60s genes were found to be upregulated (Fig. 9). The maximum upregulation was observed in OsCBP60_6 followed by OsCBP60_7.
Taken together, OsCBP60_5 and OsCBP60_9 were found to be commonly upregulated in EBR and JA treatments. Similarly OsCBP60_1, OsCBP60_2, OsCBP60_3, OsCBP60_5, OsCBP60_13 and OsCBP60_14 were found to be upregulated in both EBR and SA treatments. OsCBP60_8 was found to be commonly upregulated in SA, JA, EBR phytohormones. These data helps to interpret that EBR mediates biotic stress responses in rice by modulating the both SA and JA pathways.

Discussion
Being sessile organisms in the nature, plants are constantly experiencing the changing environmental conditions, and therefore, the life cycle of plants can be affected by many stimuli generated by abiotic and biotic environmental factors. A number of environmental stimuli often cause stress to the plants and limit plant growth and development. However, the physiology of the plants has been evolved with complex systems of signal perception and transduction networks that enable them to cope up with adverse environmental conditions. It is well-recognized that calcium mediated signaling is important for plant responses to a wide variety of stresses including pathogen attack (Dodd et  ).The growth of the rice blast fungus as well as the bacterial blight were signi cantly suppressed in oscbt-1 mutant rice plants (Koo et al., 2009). However, there is no report of involvement of any OsCBP60s in disease resistance responses. A plethora of literatures are available regarding in silico candidategene identi cation in rice using Arabidopsis gene/protein sequences as bait using homology searches (Prasad et al., 2009;Boonburapong and Buaboocha 2007). Keeping in view that some members of AtCBP60s involved in disease resistance, we made an attempt to identify the rice homologue of CBP60s and characterize the genes in relation to disease resistance in rice. In present investigation, we have identi ed 15 candidate OsCBP60s in rice. All the 15 candidate genes contains only CBD domain at N-terminus as appeared in AtCBP60s (Reddy et al., 2002;Wang et al., 2009). In silico localization studies showed that more than 50% of OsCBP60s were localized to nucleus. Our results are in accordance with AtCBP60s in which 50% of genes were localized to nucleus (data not shown). Also, the subcellular localization of AtCBP60g in transgenic Arabidopsis lines were found to be in nucleus (Wan et al., 2012), which further strengthen our studies. . The SA pathway is primarily induced by and effective in mediating resistance against biotrophic pathogens, whereas the JA pathway is primarily induced by and effective in mediating resistance against herbivores and necrotrophic pathogens (Glazebrook, 2005). Brassinosteroids (BRs) are regarded as a class of essential plant hormones that plays diverse roles in monitoring broad spectrum of plant growth, developmental processes and regulate multiple physiological functions including various biotic and abiotic stresses (Li and Chory, 1999;Sreeramulu et al., 2013). Exogenously administered BR also increased general plant resistance against pathogens (Divi and Krishna 2001a;Nakashita et al., 2003;Ali et al., 2013). BR positively regulates SA and JA pathway components NPR1 to mediate defence gene expression (Divi et al., 2010). The collective contribution and timing of these hormones during plant-pathogen interactions are crucial to determine the success of the interactions. To analyze the underlying molecular mechanisms for SA, JA and BR-induced stress tolerance, we examined the effects of these phytohormones on the expression of 15 OsCBP60s. In SA and EBR treatments, the induced expression of OsPR1a was recorded, whereas down regulation of OsPR1a was observed at 24 and 48 h of JA treatment. A plethora of reports indicates that treatment with SA up-regulates the PR1 gene expression in plants (Rivière et al., 2008) including rice (Mitsuhara et al., 2008). EBR also induced expressions of genes encoding proteins involved in defense leading to induction of PR1 (Xia et al., 2009). In our study, the induction of OsPR1a gene expression was observed in EBR treated samples, which supports the earlier reports. The upregulation of OsCBP60s in SA treated samples indicates positive regulation. The observed upregulation of OsCBP60s genes in SA treatment and downregulation in JA treatment supports the previously reported antagonistic interactions between the SA and JA mediated signaling pathways (Hu et al., 2013). The upregulation of 4 OsCBP60s were observed in both SA and JA treatment. SA and JA defense pathways generally antagonize each other but the interactions between those two pathways can be synergistic (Mur et al., 2006;Koornneef et al., 2008). All OsCBP60s were found to be upregulated in EBR treated samples, which indicates the positive regulation between EBR and OsCBP60s. In rice, BR enhances tolerance to various biotic factors including M. oryzae and X. oryzae (Ahmad et al., 2018). Taken together, these results support the complex crosstalk between different phytohormones. The upregulation OsCBP60_5 gene in pathogens, SA and EBR treatments showed its potentiality for engineering biotic stress tolerance in rice.
There is a growing realization that biotic stresses triggers Ca 2+ signaling pathways in plants.
Phytohormones as well as pathogens trigger OsCBP60s, which modulates biotic stress responses. The presence of several stresses responsive TFBSs, identi ed through In silico searches, further strengthen the role of OsCB60s in modulating stress responses in rice. Based on the results of this study, a model incorporating pathogens and phytohormones studied modulating OsCBP60s alone or via crosstalk to enhanced biotic stress in rice is depicted in Figure 10.

Figure 7
Response of 15 OsCBP60s gene in SA treated rice seedlings. Leaf samples were collected at the indicated time points. Transcript levels were analyzed by qRT-PCR analysis and expressed relative to the mock treatment at each time point. OsPR1a gene is the marker gene for SA mediated defense signaling in rice.
Results are representative of three independent experiments. Error bars represent standard error (SE) of mean for three replicates.