Revisiting the Evolution and Function of NIP2 Paralogues in the <i>Rhynchosporium</i> Spp. Complex

ABSTRACT 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 paralogues remain uncharacterised. Here we present a highly contiguous long‐read assembly of a newly isolated Australian strain, R . commune WAI453, that is virulent on multiple barley cultivars. 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 other species in the Rhynchosporium genus. Some NIP2 paralogues have subsequently been lost or are absent in these closely related species. The diversity of these paralogues was examined from R . commune global populations and their expression was analysed during in planta and in vitro growth to evaluate the importance of these genes during infection. The majority of NIP2 and NLP paralogues in the WAI453 genome were significantly upregulated during plant infection suggesting that the NIP2 and NLP genes harbour virulence roles. An attempt to further characterise the 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.