Heterologous expression and characterization of ToxA1 haplotype from India and its interaction with Tsn1 for spot blotch susceptibility in spring wheat

ToxA, a necrotrophic effector protein, is present in the genome of fungal species like Parastagnospora nodorum, Pyrenophora tritici-repentis and Bipolaris sorokiniana.Tsn1 is the sensitivity gene in the host whose presence indicates more susceptibility to ToxA carrying pathogen, and ToxA-Tsn1 interaction follows an inverse gene-for-gene relationship. The present study involved cloning and expressing the ToxA1 haplotype from B. sorokiniana. It was found that the amplicon exhibited an expected product size of 471 bp. Sequence analysis of the ToxA1 nucleotide sequence revealed the highest identity, 99.79%, with P. tritici-repentis. The protein expression analysis showed peak expression at 16.5 kDa. Phylogenetic analysis of the ToxA1 sequence from all the Bipolaris isolates formed an independent clade along with P. tritici-repentis and diverged from P. nodorum. ToxA-Tsn1 interaction was studied in 18 wheat genotypes (11 Tsn1 and 7 tsn1) at both seedling and adult stages, validating the inverse gene-for-gene relationship, as the toxin activity was highest in the K68 genotype (Tsn1) and lowest in WAMI280 (tsn1). The study indicates that the haplotype ToxA1 is prevailing in the Indian population of B. sorokiniana. It would be desirable for wheat breeders to select genotypes with tsn1 locus for making wheat resistant to spot blotch.

Gangetic plains of India. Wheat productivity can decline by 40% due to spot blotch alone (3). 59 Spot blotch has been reported to cause up to 17.5% yield loss in the Indian sub-continent, and  The ToxA protein was first discovered by Tomás and Bockus (9) from the culture filtrate of P. 69 tritici-repentis. The ToxA manifested high nucleotide diversity in P. nodorum, whereas it 70 showed a lack of variation in P. tritici-repentis. This pattern of nucleotide diversity and the 71 recent emergence of tan spot suggests the recent introduction of ToxA into P. tritici-repentis 72 genome (6). The ToxA sequences from P. nodorum and P. tritici-repentis depicted differences 73 at four fixed nucleotide sites resulting in two predicted amino acid changes consistent with the 74 interspecific transfer of this gene from S. nodorum into P. tritici-repentis (6), and this ToxA 75 gene jumped horizontally from P. tritici-repentis to another pathogen B. sorokiniana, (10). 76 ToxA and its surrounding 14 kb suggest that this gene was horizontally transferred, facilitated 77 by a type II DNA transposon (11). ToxA is a single-copy gene, and it was found to be embedded 78 within a 12-kb genomic element nearly identical to the corresponding regions in P. nodorum 79 and P. tritici-repentis (10). The mature ToxA protein is 13.2 kDa, and it encodes a signal 80 peptide of 23 amino acids and a pro-domain of 4.3 kDa (required for protein folding), and both 81 are cleaved before the secretion of mature ToxA protein (1, 12, 13). Studies revealed that the 82 host sensitivity of ToxA was mapped on wheat chromosome arm 5BL, and the sensitivity gene 83 was named Tsn1 (14). Further, it was confirmed that this gene was significant for both tan spot 84 and SNB, and in both cases, the pathogen had a sensitivity gene such as ToxA (6,15 using Nanodrop2000 instrument (Thermo Scientific) and 1µg RNA reverse transcribed to 119 cDNA was synthesized using a Thermo cDNA synthesis kit following the manufacturer's 120 protocol.

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An additional treatment of RNase was included during cDNA synthesis as supplied and 122 instructed with the kit and manufacturer's protocol to prevent chances of contamination of 123 genomic DNA during isolation.

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ToxA gene cloning 125 The expression construct was produced in the bacterial expression vector pET28a that directs     The screening of 18 wheat genotypes was conducted for Tsn1/tsn1, out of which 11 genotypes 203 harboring Tsn1 and the rest 7 genotypes with tsn1 (see Table 1 (Fig. 2).

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The phylogenetic analysis depicts the evolutionary relatedness of the ToxA1 amino acid 245 sequence with other ToxA sequences. The tree indicates that ToxA1 sequences originate from 246 a common ancestor but form different clades during evolution. The ToxA1 sequence from all 247 Bipolaris isolates formed an independent clade along with P. tritici-repentis and diverged from 248 P. nodorum. The phylogeny indicates sequence similarity among B. sorokiniana and P. tritici-249 repentis, whereas it differed from P. nodorum (Fig. 3). reliable and good quality. The quality of the resulting models was monitored with 256 PROCHECK. Ramachandran plot analysis revealed that 88.1% of the ToxA1 protein structure 257 residues fell within the most favoured regions, with a further 11.9% in the additionally allowed 258 region (Fig. 4). No residues were found in the generously allowed or disallowed regions. The  (Fig. 5). The percentage of the alpha helix was 3.39%. At the same time, antiparallel sheet 1 270 was 35.55%, antiparallel sheet 2 was 12.76%, the parallel sheet was 0.85%, the turn was 7.63%, 271 and the other was 39.83%. 273 stage 274 The bioactivity of the ToxA1 effector was studied on necrotic lesions symptoms caused due to 275 ToxA1 protein and was found to be more prominent at the adult stage. At the seedling stage,   more closely related to one another than they are to P. nodorum. This conclusion is supported 318 by the identical ToxA1 sequence shared by B. sorokiniana and P. tritici-repentis and the six-319 position difference between P. nodorum haplotypes. (Fig. 2). The sequence identity between 320 the three fungal species accounts for the common evolutionary origin of ToxA and the potential 321 of exchanging DNA between these species (10). The significance of phylogenetic studies in 322 establishing evolutionary relationships among the different pathogen races or deriving the 323 specific gene evolution between different genera is well known. The sequencing data for ToxA1 324 showed a high similarity between B. sorokiniana and P. tritici-repentis (10, 37, 38). Previous 325 studies have also suggested that the ToxA1 sequence did not differ for B. sorokiniana and P. 326 tritici-repentis but differed from the ToxA sequence of P. nodorum (10, 34, 37, 38). This 327 evolutionary relatedness of P. tritici-repentis and B. sorokiniana sequences have also been 328 reported earlier (10, 34). The earliest report suggested that ToxA has been present in P. 329 nodorum for a long time as it exhibited greater sequence diversity and was interspecifically 330 transferred to P. tritici-repentis quite later. This is evident from monomorphism or lesser 331 sequence diversity in the ToxA1 sequence from P. tritici-repentis and the emergence of tan 332 spot symptoms more recently in the near past (38).

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Further insights into the ToxA structural analysis were performed by predicting the 3D protein 334 structure using an amino acid sequence of ToxA1 (Fig.4). This is the first report for the 3D 335 protein structure ToxA1 haplotype from B. sorokiniana. The predicted protein structure was 336 similar to a single-domain protein with a β-sandwich fold of PtrToxA. This β-fold represents a 337 solvent-exposed loop containing arginyl-glycol-aspartic acid (RGD)-functionally active motif 338 (39, 40). ToxA1 has been known to resemble with fibronectin III domain which retains a 339 solvent-exposed loop containing RGD motif for cell wall-plasma membrane interaction in        b. a. Fig. 2 (a.) The multiple sequence alignments with a phylogenetic tree, Colors both on the phylogenetic tree and the branch scale represent genetic distance. ToxA sequences have been coloured by four colours shown in the 'seq' column for each nucleotide. Colour changes on the aligned sequences represent nucleotide differences. (b) Based on the number of nucleotide variations between ToxA haplotypes, a heat map was created. The relevant ToxA haplotype in the matrix is represented by each branch of the phylogenetic tree. The near ties to "dark red" and the distance ties to "white" colour. (c) Neighbor-joining (NJ) tree for ToxA haplotypes based on the nucleotide sequence differences. Coloured internal nodes represent the bootstrap confidence level.  Table1: Response of wheat genotypes (Tsn1/tsn1) in terms of leaf necrosis after infiltration of ToxA1 protein at both seedling and adult stage of wheat genotypes harboring Tsn1/tsn1 allele.