We studied the function of Arabidopsis HISTONE MONOUBIQUITINATION 1 ( HUB1 ), the E3 ligase for histone H2B, in plant defense against necrotrophic pathogens and other plant functions. HUB1 is a homolog of yeast BRE1 ( BrefaldinA Sensitivity ) and encodes C 3 HC 4 RING E3 ligase that catalyzes the monoubiquitination of histone H2B. HUB1 was identified as the potential target of BIK1 ( B.cinerea Induced Kinase1 ), a protein kinase required for resistance to necrotrophic pathogens. HUB1 is induced in response to B. cinerea in wild type plants but is constitutively expressed at a higher level in the bik1 mutant.
The role of HUB1 in disease resistance was studied using mutant alleles and over expression transgenic lines. HUB1 mutant alleles show increased susceptibility whereas its ectopic expression was sufficient for increased resistance to B. cinerea and A. brassicicola . In contrast, hub1 plants show no altered responses to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae indicating a specific role for HUB1 in resistance to necrotrophic fungi. HUB1 is induced by B. cinerea and A. brassicicola , at the site of infection, further supporting the role of HUB1 in defense. Interestingly, hub1 plants have reduced cell wall thickness compared to wild type and overexpression lines but accumulate increased callose in response to B. cinerea and A. brassisicola . Thus, HUB1 may regulate resistance by altering cell wall related defenses.
The genetic interactions between HUB1 and key regulatory genes in Arabidopsis defense responses were studied. The hub1coi1 and hub1ein2 double mutants were constructed to determine the role of jasmonate (JA) and ethylene (ET) on the functions of HUB1. ET and JA regulate Arabidopsis defense against necrotrophic fungi. The hub1coi1 and hub1ein2 double mutant shows more severe B.cinerea disease symptoms and pathogen growth than the single mutants. This, coupled with the normal expression of the JA regulated PDF1.2 gene in the hub1 mutant suggests that HUB1 functions independent of the JA and ET response pathways. The response of hub1coi1 to A. brassicicola mirrors that of B. cinerea . However, the responses of hub1ein2 to A. brassicicola suggests that EIN2 is epistatic to HUB1 for A. brassicicola resistance but is additive to HUB1 with respect to B.cinerea resistance indicative of a contrasting role for ET in resistance to these fungi.
HUB1 also regulates flowering time in Arabidopsis. The hub1 mutant flowers significantly earlier whereas the 35S: HUB1 lines flowered later than wild type under short and long day conditions. Consistent with this, the expression of MAF1 and MAF4 genes are reduced in hub1 . MAF1 and MAF4 are members of the FLC gene family that can act as floral repressors when expressed constitutively to high levels.
Lastly, HUB1 interacts with Arabidopsis MED21 subunit of mediator, an evolutionarily conserved protein complex. Arabidopsis MED21 is an essential gene as revealed from the embryo lethality of the loss of function allele. HUB1 and MED21 are both induced by chitin treatment further supporting their role in basal resistance. Importantly, our data suggest HUB1 mediated chromatin modification is linked to RNA polymerase function in transcription through MED21. In sum, histone H2B monoubiquitination is an important chromatin modification that has a regulatory role in plant defense against necrotrophic fungi most likely through modulation of gene expression. (Abstract shortened by UMI.)
