GunA of Sinorhizobium (Ensifer) fredii HH103 is a T3SS-secreted cellulase that differentially affects symbiosis with cowpea and soybean

Abstract

The symbiosis between Sinorhizobium fredii HH103 and its host legumes is influenced by the type 3 secretion system (T3SS), which delivers proteins (effectors) directly into the host cells to promote infection. GunA, one of the predicted HH103 effectors, potentially codes for a cellulase. In this work we tried to characterise GunA and elucidate its role in symbosis with soybean and cowpea. MethodsA GunA::HA fusion protein was constructed to study T3SS-dependent secretion.Cellulase activity of GunA was measured and gunA::uidA-GFP and gunA::cyA fusions were constructed to monitor gunA expression in nodules and to study translocation to the host cells, respectively. Finally, the symbiotic performance of a gunA mutant was studied in soybean and cowpea. ResultsGunA from S. fredii HH103 shows cellulase activity and is secreted through the T3SS in response to the inducer flavonoid genistein. Interestingly, at the beginning of the symbiotic process, GunA was partially responsible for the induction of the expression of the soybean GmPR1 gene, a gene used as a marker for plant defense responses.However, GunA was also detected in soybean and cowpea developed nodules. Finally, nodulation assays indicate that GunA is beneficial for symbiosis with soybean but detrimental with cowpea. Conclusion

Structural and enzymatic characterisation of the Type III effector NopAA (=GunA) from Sinorhizobium fredii USDA257 reveals a Xyloglucan hydrolase activity

“…NopAA from Sinorhizobium fredii USD257 has Xyloglucanase activity. NopAA analogs have been shown to have cellulase activity revealed with CMC-gelose assay combined with congo-red staining 22,23 . As NopAAΔ48 shows a cellulase activity analog to NopAA using this method ( Supplementary Fig.…”

“…enzymatic activity assays. Preliminary carboxymethylcellulose (CMC) gelose assay combined with congo-red staining were performed using the protocol described in 23 . To determine the specificity of NopAAΔ48, we monitored during one hour the degradation of the following substrates: carboxymethylcellulose 4 M (CMC), mixed-linked glucan (MLG, also referred to as β-glucan from barley, which contains a ratio of 1/4 β-1,3/β-1,4 linkages), tamarind xyloglucan (Xlg), glucomannan from konjac (Glm, which contains a ratio of 3/1 mannose/ (2020) 10:9932 | https://doi.org/10.1038/s41598-020-67069-4 www.nature.com/scientificreports www.nature.com/scientificreports/ glucose units), and laminarin (Lam).…”

“…Recombinant NopAA from B. diazoefficiens USDA 110 was shown to have cellulase activity 22 , although it is not crucial for the nodulation process. In Sinorhizobium fredii HH103, GunA has been shown to possess also a cellulase activity but it also plays an important role in host specificity as its inactivation differently affects symbiosis with soybean and with cowpea 23 .…”

QTL mapping and gene mining to identify genes on soybean (Glycine max) associated with NopB of Sinorhizobium fredii HH103

“…At least 12 effectors [NopA, NopB, NopX, NopM, NopL, NopP, NopT, NopC, NopE, NopJ, NopAA (GunA) and NopI (Bartsev, Boukli, Deakin, Staehelin, & Broughton, ; Dai, Zeng, Xie, & Staehelin, ; Deakin, Marie, Saad, Krishnan, & Broughton, ; Jiménez‐Guerrero, Pérez‐Montaño, Medina, Ollero, & López‐Baena, , ; Kimbrel et al, ; Lorio, Kim, & Krishnan, ; Rodrigues et al, ; Saad, Staehelin, Broughton, & Deakin, ; Schechter, Guenther, Olcay, Jang, & Krishnan, ; Wenzel, Friedrich, Göttfert, & Zehner, ; Xin et al, )] have been identified in rhizobia. Moreover, at least eight effectors [NopA, NopX, NopM, NopL, NopP, NopD, NopC and NopI (Bellato, Krishnan, Cubo, Temprano, & Pueppke, ; Kambara et al, ; Kimbrel et al, ; Krishnan, Kuo, & Pueppke, ; López‐Baena et al, ; Lopez‐Baena et al, ; de Lyra et al, ; Rodrigues et al, ; Thomas, ; Viprey, Greco, Golinowski, Broughton, & Perret, )] have been identified in Sinorhizobium fredii HH103 (Jiménez‐Guerrero et al, ). Among these Nops, NopA and NopX are important structural components of the T3SS pili, and both are necessary for the secretion of other Nops (Saad et al, ).…”

“…Among these Nops, NopA and NopX are important structural components of the T3SS pili, and both are necessary for the secretion of other Nops (Saad et al, ). NopAA (GunA) is a cellulase that can break down the host legume cell wall to promote rhizobia infection (Jiménez‐Guerrero et al, ). NopC can be secreted into soybean root cells and has a positive function during infection (Jiménez‐Guerrero, Pérez‐Montaño, Medina, Ollero, & López‐Baena, ).…”