Revisiting the evolution and function of NIP2 parologs in the Rhynchosporium spp. complex

The fungus Rhynchosporium commune, the causal agent of barley scald disease, contains a paralogous effector gene family called Necrosis-Inducing Protein 2 (NIP2) and NIP2-like protein (NLP). However, the function and full genomic context of these paralogs remains uncharacterised. Here we present a highly contiguous long-read assembly of R. commune WAI453. Using this assembly, we show that the duplication of the NIP2 and NLP gene families is distributed throughout the genome and pre-dates the speciation of R. commune from its sister species. Some NIP2 paralogs have subsequently been lost or are absent in the sister species. The diversity of these paralogs was examined from R. commune global populations and their expression was analysed during in planta and in vitro growth to analyse the importance of these genes during infection. The majority of NIP2 and NLP paralogs in WAI453 genome were significantly upregulated during plant infection suggesting that the NIP2 and NLP genes harbour virulence roles. An attempt to further characterise function of NIP2.1 by infiltrating purified protein into barley leaves did not induce necrosis questioning its previously reported role as an inducer of host cell death. Together these results suggest that the NIP2 effector family does play a role during infection of barley, however the exact function of NIP2, like many effectors, remains uncharacterised.