Does plant –microbe’s common Inter Simple Sequence Repeat lead to a new recombination? A novel mechanism of pathogenicity

Abstract A total of eight varieties of the mango from an orchard were studied using molecular markers to understand the host-pathogen interaction. From the infected leaves of the plant, a total of the 8 bacterial pathogens (Exiguobacterium arabatum, Pseudomonas mendocina, Pantoea dispersa, Bacillus sp. Pantoea ananatis, Micrococcous luteus, Microbacterium_sp., Enterobacter cloacae) were isolated and identified. All the host varieties of mango were distinguished for the genetic diversity using the Inter Simple Sequence Repeat (ISSR) DNA markers. This set of ISSR marker primers were also used for the mango pathogens. PCR amplification of the ISSR primers showed polymorphic and monomorphic band patterns in the host plants and in their pathogens. The monomorphic band generated by PCR amplification in the host and in the pathogen, by the common primer, is selected and used for PCR hybridization technique. PCR products obtained from the host, pathogen and hybridization were cloned, sequenced and compared. A multiple sequence alignment of these sequences revealed that the product of hybridization PCR was mixture of host and pathogen sequences. On this basis, we hypothesize a possibility for the recombination of host-microbes DNA as one of the mechanisms of pathogenicity for the plant pathogens using hybrid PCR technique. The possible mechanism of recombination for plant host and its pathogen is discussed. Highlights Inter Simple Sequence Repeat markers used to (i) Fingerprint the pathogens and their host (mango) and (ii) for study of the possibilities for the recombination as mechanism of pathogenicity.


53
Mechanisms of pathogenicity of phytopathogens are being extensively studied in  negative and some Gram-positive bacteria (Baker et al., 2010;Venturi and Fuqua, 2013). 55 These studies revealed that pathogens contain suits of genes including various transporter 56 systems (Chen et al., 2010;Rakhashiya et al., 2016;Wilkens, 2015), secretion systems 57 (TSS), virulence and associated metabolism (Abby et al., 2016;Chang et al., 2014;Green 58 and Mecsas, 2016). In pathogens, from the cytoplasm through the inner membrane or the outer 59 membrane to the extracellular space, the secretion of proteins requires dedicated types of 60 machinery, i.e. type I to V secretion pathways (Thanassi and Hultgren, 2000). The distinction in 61 these pathways from each other is mainly by the presence or absence of a signal peptide on the 62 secreted protein and by the mode of translocation steps. Using these types of machinery, 63 pathogen gets an entry into the host cell and colonizes there. We report in this study, a probable  Many fruit species are screened for the desired characters with the molecular markers such as 71 banana (Brown et al., 2009), litchi (Chundet et al., 2007, pummel (Uzun et al., 2013), grape 72 (Vafaee et al., 2017) and sweet orange (Dehesdtani et al., 2007). Inter-simple sequence 73 repeats (ISSRs) is a PCR-based DNA marker technique shows reproducible results and it is 74 widely used for the identification of plant and animal species but the references in 75 bacteriology are rather scanty. It is found in the genome flanked by microsatellite sequences 76 (Simple Sequence Repeat) regions. Random DNA segments in the genome can be PCR-77 amplified by using arbitrarily designed primers. Such experiments usually produce multiple 78 DNA fragments (each of which is considered a locus) in a single reaction. Thus without the 79 need to first know the DNA sequences of the target regions, allowing the generation of a 80 large number of loci across the genome of any species. The technique is mainly useful for the 81 understanding evaluation of genetic diversity, cultivar identification and phylogenetic 82 relationship of the studied organisms. The simplicity of ISSR markers predetermines them for 83 gene tagging and it is also used with QTL analysis (Agarwal et al., 2008;Khaled and 84 Hamam, 2015;Malik et al., 2014). ISSR with its ease of application, we envision that it can 85 be explored to comprehend the host-microbe interaction.

87
Mango (Mangifera indica L.), 'the king of the fruits' is an economically important plant with 88 high nutritional value (Sivakumar et al., 2011;Tharanathan et al., 2006). Nine different host 89 varieties of mango from the orchard are studied for the ISSR primers. The same primers are   (Table 1).

128
DNA of pathogens and/or host plant varieties was subjected to ISSR primer amplification. A 129 set of 30 different ISSR primers were used for this study ( Table 2). The total volume of 25 µl

Cloning
The amplified products of first PCR (pathogen and host) and second PCR (hybrid) were 147 cloned and sequenced. UBC 814 primer generated monomorphic bands were cloned by using 148 'InsTAclone PCR Cloning Kit' as suggested by the manufacturer (Thermo Scientific, USA).

149
As per the protocol, direct one-step cloning using TA system for PCR products with 3'-dA 150 overhangs to dT overhangs of vector was done. E. coli strain DH5α with lacZΔM15 mutation 151 was used for the transformation of cloned vectors. The plates were incubated for 16 hours at 152 37°C temperature and screened for blue/white colonies to check successful transformation.

153
The white colonies were selected for the plasmid isolation by alkaline lysis method 154 (Sambrook et al., 1989) .  all the organisms showed at least one common ISSR primer amplification with the 9 th variety 202 (Fig. 1). Each ISSR bands were scored as present (1)  Totapuri), for the hybridization assay as both generate monomorphic band with ISSR primer 214 UBC814 (Fig. 3). The hybrid extension by PCR as used in the present study involves three 215 separate PCRs; the two DNA fragments produced in the ISSR reactions in bacteria and plant, 216 and the mixture to form the hybrid template for the next stage. It was assumed that hybrid 217 template will have some primer binding sites common to plant and bacteria and the middle 218 portion of the sequences may/ may not be similar and subsequent PCR should generate 219 recombinant DNA sequences. ISSR marker (UBC 814) gave monomorphic band patterns in 220 the results (Fig.2); which can be cloned and used as a molecular marker for the individual 221 pathogen/plant. In addition, the primer generates monomorphic band in host and pathogen is 222 tested for the hybrid PCR and sequencing. For this, the monomorphic bands were taken and 223 denatured at 95˚C in PCR and allowed to cool down at room temperature. In fact, the 224 polymorphic bands generated more opportunities for recombination of two different DNAs; 225 but here selected monomorphic band, considering the ease of the assay to understand the 226 mechanism. The PCR mixture used as template DNA with the same primer set was amplified 227 and sequenced (Fig. 4). Further, the hybrid PCR with the DNA templates of pathogen and 228 host generated chimeric DNA which might have joined in the PCR cycles within short 229 stretches of identity. The characteristic of the end products is an important concern in 230 understanding recombination reactions; for example, are their overall structures simply or 231 multiply exchanged? Further, is the joining homologous or illegitimate? The newly formed 232 sequence is accurate or mutagenic? In this deference, the most important feature of the newly 233 generated sequence is that either template is applied for the point of new recombination ( It revealed that sequence of plant and pathogen are different in alignment results; but the 240 hybrid sequence showed a mixture of both, randomly. We proposed that the splicing overlap 241 and mutations in the hybrid sequence observed attributed to the ISSR technique that uses 242 single primer for forward and reverse amplification. We also observed that the length of the 243 monomorphic band of host and pathogen were not exactly equal and that each PCR cycle increased the probabilities of variations (Fig.3, 4). Such these results suggest that the 245 possibilities of recombination in host-pathogen cannot be ruled out.

Conflict of Interest 251
The authors declare no conflict of interest.