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Co-infection, caused by multiple pathogen attacks on an organism, can lead to disease development or immunity. This complex interaction can be synergetic, co-existing, or antagonistic, ultimately influencing disease severity. The interaction between fungus, bacterium, and virus (three kingdom pathogens) is most prevalent. However, the underlying mechanisms of co-infection need to be explored further. In this study, we investigated the co-infection phenomenon in rice plants exposed to multiple pathogen species, specifically Rice necrosis mosaic virus (RNMV) and rice blast fungus (Magnaporthe oryzae, MO), bacterial leaf blight (Xanthomonas oryzae pv. oryzae, XO) or Cucumber mosaic virus (CMV). Our research showed that RNMV interacts synergistically with MO, XO, or CMV, increasing pathogen growth and lesion size. These findings suggest positive synergy in RNMV co-infections with three kingdom pathogens, increasing accumulation and symptoms. Additionally, to investigate the role of RNAi in pathogen synergism, we analyzed rice mutant lines deficient in RNA-dependent RNA polymerase 1 (OsRDR1) or 6 (OsRDR6). Notably, we observed the loss of synergy in each mutant line, highlighting the crucial role of OsRDR1 and OsRDR6 in maintaining the positive interaction between RNMV and three kingdom pathogens. Hence, our study emphasized the role of the RNA silencing pathway in the intricate landscape of pathogen interactions; the study's outcome could be applied to understand the plant defense response to improve crop yields., Competing Interests: Declaration of Competing Interest All the authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the Arabidopsis Multi-Antibiotic Resistance 1 ( MAR1 ) /RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium- mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacum MAR1 homologs ( NtMAR1h s). All three sgRNAs directed the introduction of InDels into NtMAR1h s-knockout seedlings of the T NtMAR1h s. The NtMAR1h s-knockout seedlings of the T 1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1 , NtMAR1h s have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.