The harpin protein HrpN_Ea induces Arabidopsis resistance to the green peach aphid by activating the ethylene signalling pathway and by recruiting EIN2, an essential regulator of ethylene signalling, for a defence response in the plant. We investigated 37 ethylene-inducible Arabidopsis transcription factor genes for their effects on the activation of ethylene signalling and insect defence. Twenty-eight of the 37 genes responded to both ethylene and HrpN_Ea, and showed either increased or inhibited transcription, while 18 genes showed increased transcription not only by ethylene but also by HrpN_Ea. In response to HrpN_Ea, transcription levels of 22 genes increased, with AtMYB44 being the most inducible, six genes had decreased transcript levels, and nine remained unchanged. When Arabidopsis mutants previously generated by mutagenicity at the 37 genes were surveyed, 24 mutants were similar to the wild type plant while four mutants were more resistant and nine mutants were more susceptible than wild type to aphid infestation. Aphid-susceptible mutants showed a greater susceptibility for atmyb15, atmyb38 and atmyb44, which were generated previously by T-DNA insertion into the exon region of AtMYB15 and the promoter regions of AtMYB38 and AtMYB44. The atmyb44 mutant was the most susceptible to aphid infestation and most compromised in induced resistance. Resistance accompanied the expression of PDF1.2, an ethylene signalling marker gene that requires EIN2 for transcription in wild type but not in atmyb15, atmyb38, and atmyb44, suggesting a disruption of ethylene signalling in the mutants. However, only atmyb44 incurred an abrogation in induced EIN2 expression, suggesting a close relationship between AtMYB44 and EIN2.

In Arabidopsis thaliana (Arabidopsis) treated with the harpin protein HrpN_Ea, resistance to the green peach aphid Myzus persicae, a generalist phloem-feeding insect, develops with induced expression of the AtMYB44 gene. Special GLUCAN SYNTHESIS-LIKE (GSL) genes and 𝛽-1,3-glucan callose play an important role in plant defence responses to attacks by phloem-feeding insects. Here we report that AtGLS5 and AtMYB44 are both required for HrpN_Ea-induced repression of M. persicae feeding from the phloem of Arabidopsis leaves. In 24 h successive surveys on large-scale aphid populations, the proportion of feeding aphids was much smaller in HrpN_Ea-treated plants than in control plants, and aphids preferred to feed from the 37 tested atgsl mutants rather than the wild-type plant. The atgsl mutants were generated previously by mutagenesis in 12 identified AtGSL genes (AtGSL1 through AtGSL12); in the 24 h survey, both atgsl5 and atgsl6 tolerated aphid feeding, and atgsl5 was the most tolerant. Consistently, atgsl5 was also most inhibitive to the deterrent effect of HrpN_Ea on the phloem-feeding activity of aphids as monitored by the electrical penetration graph technique. These results suggested an important role of the AtGSL5 gene in the effect of HrpN_Ea. In response to HrpN_Ea, AtGSL5 expression and callose deposition were induced in the wild-type plant but not in atgsl5. In response to HrpN_Ea, moreover, the AtMYB44 gene known to be required for repression of aphid reproduction on the plant was also required for repression of the phloem-feeding activity. Small amounts of the AtGSL5 transcript and callose deposition were detected in the atmyb44 mutant, as in atgsl5. Both mutants performed similarly in tolerating the phloem-feeding activity and impairing the deterrent effect of HrpN_Ea, suggesting that AtGSL5 and AtMYB44 both contributed to the effect.

Hpa1 is a harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpa1, we generated an inactive variant protein, Hpa1𝛥NT, by deleting the nitroxyl-terminal region of the Hpa1 sequence and compared Hpa1𝛥NT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpa1 was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes in plant leaves. The growth-promoting activity of Hpa1 was further correlated with a physiological consequence shown as promoted leaf photosynthesis as a result of facilitated CO₂ conduction through leaf stomata and mesophyll cells. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the Hpa1𝛥NT treatment. These analyses suggest that Hpa1 requires the nitroxyl-terminus to facilitate CO₂ transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant.

Autophagy is a highly conserved intracellular degradation pathway in eukaryotic cells that responds to environmentalchanges. Genetic analyses have shown that more than 40 autophagy-related genes (ATG) are directly involved in thisprocess in fungi. In addition to Atg proteins, most vesicle transport regulators are also essential for each step of autophagy.The present study showed that one Endoplasmic Reticulum protein in Saccharomyces cerevisiae, Tip20, which controlsGolgi-to-ER retrograde transport, was also required for starvation-induced autophagy under high temperature stress. Intip20 conditional mutant yeast, the transport of Atg8 was impaired during starvation, resulting in multiple Atg8 punctadispersed outside the vacuole that could not be transported to the pre-autophagosomal structure/phagophore assembly site(PAS). Several Atg8 puncta were trapped in ER exit sites (ERES). Moreover, the GFP-Atg8 protease protection assayindicated that Tip20 functions before autophagosome closure. Furthermore, genetic studies showed that Tip20 functionsdownstream of Atg5 and upstream of Atg1, Atg9 and Atg14 in the autophagy pathway. The present data show that Tip20,as a vesicle transport regulator, has novel roles in autophagy.